Fluorescent dye binding peptides

ABSTRACT

The present invention is directed to novel polypeptides, termed fluorettes, that bind with high avidity to fluorophore dyes. The peptides find use in a variety of methods and approaches involving fluorophore dyes.

This application is a continuation-in-part of application Ser. No.60/104,465 filed Oct. 16, 1998.

FIELD OF THE INVENTION

The invention relates to peptides that bind to fluorescent dyes, termed“fluorettes”, and to methods of making and using the fluorettes. Inparticular, the fluorettes can be used in detection and assay systems invitro and in vivo.

BACKGROUND OF THE INVENTION

Fluorophore dyes, due to their exquisite sensitivity and ease of use,are widely used in numerous approaches in fluorescent microscopy, flowcytometry and other detection systems (Haugland. Handbook of fluorescentprobes and research chemicals (sixth edition). Molecular Probes, Inc.,Eugene Oreg. (1996) and ref. therein).

Detection of proteins in living cells using fluorescence approaches hasbeen accomplished in a variety of settings. For instance, it is possibleto use ligands (or naturally-derived antibodies) conjugated directly orindirectly to fluorophores as probes of the expression levels of nearlyany given surface-expressed protein on living cells. In some particularcases for proteins within cells, it is possible to use permeable ligandsfor individual target proteins. In these cases the ligand is eitherself-fluorescent, becomes fluorescent upon binding or is conjugated tofluorescent adducts. In other cases, it has been possible for many yearsto genetically fuse reporter enzymes such as β-galactosidase,β-glucuronidase, and β-glucosidase to proteins and use a fluorogenicdye, acted upon by the reporter enzyme(s), to assay enzymatic activityon a cell by cell basis (Nolan et al. Proc. Natl. Acad. Sci. U.S.A.85:2603-2607 (1988); Lorincz et al. Cytometry 24:321-329 (1996); Krasnowet al. Science. 251:81-85 (1991). Other systems, including β-lactamase(Zlokarnik et al. Science 279:84-88(1998)) build upon those findings byapplying dyes with increased cell permeability or having radiometricfluorescent qualities that might have advantages in some applications.In recent years proteins with inherent fluorescence, such as GreenFluorescent Protein (Welsh and Kay. Curr. Opin. Biotechnol. 8:617-622(1997); Misteli and Spector. Nat. Biotechnol. 15:961-964 (1997)) havebecome widespread in their application owing to ease of use, theavailability of mutant proteins with differing spectral qualities ineither excitation or emission, and the relative non-toxicity of theapproach.

However, in the aforementioned cases the approaches are limited by aneed to genetically fuse a relatively bulky reporter protein to themolecule under study. This can have detrimental consequences to thefunctionality of the protein in question or interfere mechanisticallywith cellular constituents with which the protein interacts. While itwould be best to directly measure a given target protein using aspecific fluorescent dye that recognized any given target moiety, notechnology exists as yet to create such reagents.

There is a need, therefore, to develop approaches that provide thebuilding blocks for specific biomaterial detection.

SUMMARY OF THE INVENTION

The invention provides peptides that bind to fluorophore dyes. In oneaspect of the invention the peptides are made of naturally occuringamino acids, non-naturally occurring amino acids, or combinationsthereof.

In another aspect of the invention, methods are provided for isolatingand identifying peptides that bind to fluorophore dyes. The methodcomprises creating and screening peptide libraries that bind tofluorophores.

In another aspect of the invention, methods are provided for increasingthe binding affinity of the fluorette for a fluorophore.

In a further aspect of the invention are provided complexes offluorettes bound to fluorophore dyes. The binding of the fluorophore bythe fluorette may alter the excitation and/or the emission spectrum ofthe fluorophore.

In an additional aspect, the present invention provides methods fordetecting a fluorette by binding a fluorette to a fluorophore dye anddetecting the fluorette/fluorophore dye complex.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Fluorophore dye carriers. Fluorescein, Oregon Green 514,Rhodamine Red and Texas Red activated derivatives were covalently linkedto the polymer carrier Ultralink Immobilized DADPA via a 12-atomdiaminodipropylamine spacer. A spacer is shown as a horizontal thickbar. Digits with arrows in the chemical structures of all fluorophoredyes, except Oregon Green 514, show that “mixed isomers” of thesefluorophore dye activated derivatives were used for a coupling.

FIG. 2. Biopanning with a peptide phage display library againstFluorescein, Oregon Green 514, Rhodamine Red and Texas Red carriers. APh.D.-12 phage display peptide library (New England Biolabs) is acombinatorial library of 12-mer peptides fused to the amino-terminus ofa minor coat pIII protein of the bacteriophage M13mp19. Bound phage infour biopanning rounds were calculated as a percentage of eluted phageplaque forming unit (plaque forming unit (pfu)) yield vs input phageplaque forming unit (pfu). All experiments were performed in duplicate.Standard deviations did not exceed 19%.

FIG. 3. Phage-fluorophore dye binding. Fluorophore dye-specific OG403,OG402, RhR401 and TR401 phage were bound to Oregon Green 514,Fluorescein, Rhodamine Red and Texas Red “free” dyes, respectively (seetext). The normalized particle amounts of amplified Ph.D.-12 phagedisplay peptide library (nonspecific phage) were bound in solution withthe same dyes as controls. Phage-fluorophore dye complexes wereprecipitated three times by PEG to remove unbound dye and spotted to anitrocellulose filter. The filter was scanned on the Storm 840 scanner(Molecular Dynamics). The presented data is from a single scan and imageenhancements were done simultaneously on the complete set of dye bindingresults using NIH Image 1.59 software. Specific/nonspecific signalratios were quantified by densitometry of spot images using NIH Image1.59 software (see Materials and Methods for details). All bindingexperiments shown were completed in duplicate. Densitometry was carriedout prior to contrast enhancement. Brightness and contrast values weremodified to 50 and 65, respectively in Adobe Photoshop. Individualelements of the figure were arranged as a composite from the sameoriginal scan from which the binding ratios were determined.

FIG. 4. Biopanning with four biased combinatorial phage display peptidelibraries based on degenerated fluorettes against Fluorescein, OregonGreen 514, Rhodamine Red and Texas Red carriers. OG402-91 CL,OG403-91CL, RhR401-91CL and TR401-91CL biased combinatorial phagedisplay peptide libraries (see text) were subjected to a biopanningagainst Fluorescein, Oregon Green 514, Rhodamine Red and Texas Redcarriers, respectively. Bound phage in three biopanning rounds werecalculated as described in a legend to FIG. 2. All experiments wereperformed in duplicate. Standard deviations did not exceed 21%.

FIGS. 5A and 5B. Peptide—Texas Red binding. Excitation and emissionspectra. Peptides at the noted concentrations were incubated with TexasRed (50 nM), or without, in 0.6 ml of TBS for 1 hr at room temperature(RT). Samples were scanned on a spectrofluorimeter (SPEX Fluoromax(Jobin Yvon-SPEX Instruments Co.)). Peak excitation or emissionpositions are shown in the figure using vertical bars. Excitation andexcitation/emission peak shifts for PepTR401(10 μM)/Texas Red andPepTRS311 (10 μM)/Texas Red complexes, respectively (A), werereproducible in three independent experiments. The results of a singleexperiment are presented.

FIG. 6. Texas Red-specific peptide PepTR401 (Panel A), PepTRS311 (PanelB), PepTR406 (Panel C) and nonspecific PepControl (Panel D) (see peptidesequences in Table 4) were bound via His₆ tag to cobalt ion-containingSepharose beads. The peptide coated beats were washed and incubated with0.5 micromolar Texas Red in TBS for 1 hour at room temperature followedby several washings of beads in order to remove unbound dye. Fluorescentand nonflourescent control beads were photographed on fluorescentmicroscope Axiophot (Zeiss) using Rhodamine Red/Texas Red filter withthe same time exposure for every sample. All binding experiments wereaccomplished in three parallels and results of a single experiments areshown.

FIGS. 7A-D. Peptide—Texas Red binding. Texas Red-specific peptides TR401(linear) (FIG. 7B) (SEQ ID NO:41), TRP501 (SKVILFE-flanked) (FIG. 7C)(SEQ ID NO:120), TRP512 (SKVILFE-flanked) (FIG. 7D) (SEQ ID NO:121), andnonspecific peptide (SKVILFE-flanked) (FIG. 7A) (SEQ ID NO:122) as anegative control were bound via polyhistidine (His₆) tag to cobaltion-containing Sepharose beads. The peptide-coated beads were washed andincubated with 0.5 μM Texas Red in TBS buffer for 1 hour at roomtemperature followed by several washings of beads in order to removeunbound dye. Fluorescent and nonfluorescent control beads werephotographed on fluorescent microscope Axiophot (Zeiss) using RhodamineRed/Texas Red filter with the same time exposure for every sample. Allbinding experiments were accomplished in duplicate and results of asingle experiment are shown.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel peptides or “fluorettes” thatspecifically bind fluorophore dyes. The present invention also providesnovel compositions comprising multimeric fluorettes and fluorettes fusedor linked to other compounds or molecules. Also provided are methods tocreate and modify fluorettes.

The present invention further provides methods for the use of fluorettesin detecting biological materials, molecules, or target analytes,intracellular events, and intermolecular and intramolecularinteractions. The fluorettes also find use in in vitro assays and highthroughput screens. The methods provided are based on the formation anddetection of a fluorette/fluorophore dye complex.

The compositions and methods of the present invention provide asignificant improvement over conventional light-emitting techniques.Fluorettes, due to their small size, are not intrusive to the systemsbeing studied and, therefore, permit detection and analysis of a targetmoiety or molecule while minimizing target modification. Currentlight-emitting detection methods involve the use of enzyme-generatedfluorochromes, luciferase-generated light, or reporter systems usingengineered, inherently fluorescent proteins such as Aequoria victoriagreen fluorescent protein (GFP). These systems are unwieldy and bulkyand are intrusive to the system being studied; often necessitating thesynthesis of large fusion proteins or out of context genomicconstructions. Thus, conventional techniques can involve substantialgenetic manipulations of the targets one is trying to study, which maydisrupt, interfere, or alter the process being measured. Fluorettesminimize or avoid these limitations.

Accordingly, the present invention provides a peptide, sometimes termeda “fluorette” herein, that will bind a fluorescent dye.

By “peptide” or “fluorette” herein is meant at least about 8 covalentlyattached amino acids. The peptide may be made up of naturally occurringamino acids and peptide bonds, or synthetic. peptidomimetic structures.Thus “amino acid”, or “peptide residue”, as used herein means bothnaturally occurring and synthetic amino acids. For example,homo-phenylalanine, citrulline and noreleucine are considered aminoacids for the purposes of the invention. “Amino acid” also includesimino acid residues such as proline and hydroxyproline. The side chainsmay be in either the (R) or the (S) configuration. In the preferredembodiment, the amino acids are in the (S) or L-configuration. Ifnon-naturally occurring side chains are used, non-amino acidsubstituents may be used, for example to prevent or retard in vivodegradations. The peptides can be linear or branched. Amino acidsincludes naturally occurring amino acids or non-naturally occurringamino acids.

By “naturally-occurring amino acids” herein is meant amino acids thatare produced by living organisms. Naturally-occurring amino acids arepreferred in in vivo embodiments.

By “non-naturally-occurring amino acids” herein is meant amino acids notproduced by a living organism but that can be chemically synthesized.Non-naturally-occurring amino acids include, for example, stereoisomersor enantiomers of the naturally-occurring amino acids in which the aminoside chains, are in either the (D)((R)) or (S)((L)) configuration; aminoacids in which an amine group is bonded to any other but thealpha-carbon or may have more than one amine group bonded to thealpha-carbon; and amino acids having functional groups or “R” groups notknown to occur in nature.

The length of the peptide or fluorette, i.e., the number of amino acids,will vary. In general, the number of amino acids varies from about 8 toabout 50, with from about 8 to about 40 being preferred, from about 8 toabout 25 being particularly preferred, and from about 8 to about 12being especially preferred. Thus, “peptide” includes peptides,oligopeptides, and in some cases, proteins.

The fluorette can be monomeric or multimeric. That is, a monomericfluorette binds a single fluorophore. A multimeric fluorette is two ormore associated monomeric fluorettes. The association of a multimericfluorette can be either covalent or non-covalent. For example, themonomers can be joined directly together, for example as a linear fusion(i.e., the carboxy terminus of one is joined to the amino terminus ofthe second), or as a branched fusion, wherein the attachment of thesecond monomer is other than to the backbone of the first. In addition,a chemical cross-linker or inclusion of specific reactive group on afluorette be used to join fluorette monomers. Alternatively themonomers, may be non-covalently associated; for example, as is morefully outlined below, dimerization sequences can be used to associatetwo monomers.

A multimeric fluorette can be homomultimeric, i.e., all the monomers arethe same, and bind the same fluorophore dye, or heteromultimeric, havingat least two different fluorette monomers and can bind differentfluorophores.

The fluorette peptides bind to at least one fluorophore dye.

By “binding” herein generally is meant a non-covalent association orinteraction between a fluorette and a fluorescent dye. The non-covalentinteractions between the fluorette and dye may involve various types ofelectrostatic, hydrophilic, and hydrophobic interactions. Binding mayalso involve forming one-or more covalent bonds between the fluoretteand the dye. Covalent bonds can be formed directly between the fluoretteand the dye or can be formed by a cross linker or by inclusion of aspecific reactive group on either the fluorette or dye or bothmolecules. Binding may also involve a combination of covalent andnon-covalent interactions.

In a preferred embodiment, the binding is specific. By “specificbinding” herein is meant that the fluorophores will preferentially bindto a fluorophore dye with a binding constant in the range of at leastabout 10^(—6) M⁻¹ to about 10⁻¹⁰ M⁻¹, with a preferred range being fromat least about 10⁻⁶ M⁻¹ to about 10^(—7) M⁻¹, with an especiallypreferred range of from at least about 10⁻⁷ M⁻¹ to about 10⁻¹⁰ M⁻¹. In apreferred embodiment, the fluorettes do not specifically bind to otherfluorophore dyes or compounds or moieties; that is, a fluorette isspecific to one fluorophore. Alternatively, a fluorette may bind two ormore fluorescent dyes (bind together 2 or more fluorophores or more orcapable of binding independently two or more fluorophores); that is, afluorette may specifically bind another compound or moiety, includinganother fluorophore, if these molecules have a common structuralfeature(s) that specifically interact with the fluorette. Preferably,however, a fluorette will not appreciably bind compounds other thanfluorescent dyes.

In other embodiments, a dye is non-fluorescent but becomes fluorescentwhen bound by a fluorette. In further embodiments, fluorette binding toa non-fluorescent dye causes the release of a fluorescent moiety. Whenthe fluorescent moiety is released, the fluorette may bind to thereleased fluorescent moiety, may bind to the non-fluorescent dye, or maybe released and therefore free to bind a second molecule of thenon-fluorescent dye and repeat this process.

By “fluorescent dye” or “fluorophore” or “fluorophore dye” herein ismeant a compound that absorbs an incident light of a characteristicrange of wavelengths or excitation spectrum and dissipates the absorbedenergy by emitting light of a characteristic range of wavelengths oremission spectrum. By “excitation spectrum” herein is meant thewavelength of incident light absorbed by the fluorophore dye that causesthe fluorophore dye to fluoresce. By “emission spectrum” herein is meantthe characteristic wavelengths of the emitted or fluorescent lightproduced as the energy of the absorbed incident light is released. Theexcitation and emission spectra for a fluorophore dye may or may notoverlap. In a preferred embodiment the incident light is in theultraviolet spectrum and the emitted light is in the visible spectrum.

Preferred fluorophores include, but are not limited to, Texas Red,Rhodamine Red, Oregon Green 514, and Fluorescein. Examples offluorescent dyes are found in the Molecular Probes Catalog, 6th Ed.,Richard Haugland, Ed., which is expressly incorporated by reference inits entirety.

In addition, the fluorette can further comprise additional components,such as a fusion partner or functional group. By “fusion partner” or“functional group” herein is meant a sequence that is associated withthe fluorettes, that confers upon the fluorette a function or ability.Fusion partners can be heterologous (i.e. not native to a host cell), orsynthetic (not native to any cell). Suitable fusion partners include,but are not limited to: a) presentation structures, as defined below,which provide the fluorettes in a conformationally restricted or stableform; b) targeting sequences, defined below, which allow thelocalization of the flourette into a subcellular or extracellularcompartment; c) rescue sequences as defined below, which allows thepurification or isolation of the fluorette; d) stability sequences,which confer stability or protection from degradation to the fluorette,for example resistance to proteolytic degradation; e) dimerizationsequences, to allow for fluorette dimerization or multimerization; or f)any combination of a), b), c), d), and e), as well as linker sequencesas needed.

In a preferred embodiment, the fusion partner is a presentationstructure. By “presentation structure” or grammatical equivalents hereinis meant a sequence, which, when fused to the fluorette, causes thefluorette to assume a conformationally restricted form. Proteinsinteract with other proteins and molecules largely throughconformationally constrained domains. Although small peptides withfreely rotating amino- and carboxy-termini can have potent functions asis known in the art, the conversion of such peptide structures intoactive agents can be difficult due to the inability to predictside-chain positions for peptidomimetic synthesis. Therefore thepresentation of fluorettes in conformationally constrained structureswill likely lead to higher affinity interactions of the fluorette withits target fluorophore dye. This fact has been recognized in thecombinatorial library generation systems using biologically generatedshort peptides in bacterial phage systems. A number of workers haveconstructed small domain molecules in which one might presentbiased-combinatorial libraries of peptide structures.

While the fluorettes are peptides, presentation structures arepreferably peptides or proteins. Thus, synthetic presentationstructures, i.e. artificial polypeptides, are capable of presentingfluorettes as a conformationally-restricted domain. Generally suchpresentation structures comprise a first portion joined to theN-terminal end of the flourette, and a second portion joined to theC-terminal end of the flourette; that is, the flourette is inserted intothe presentation structure, although variations may be made, as outlinedbelow. To increase the affinity or specificity of the fluorette, thepresentation structures are selected or designed to have minimalbiologically activity when expressed in the target cell.

Preferred presentation structures maximize accessibility to theflourette by presenting it on an exterior loop. Accordingly, suitablepresentation structures include, but are not limited to, minibodystructures, loops on beta-sheet turns and coiled-coil stem structures inwhich residues not critical to structure are randomized, zinc-fingerdomains, cysteine-linked (disulfide) structures, transglutaminase linkedstructures, cyclic peptides, B-loop structures, helical barrels orbundles, leucine zipper motifs, etc. In addition, presentationstructures include dimerization sequences, as defined below.

In a preferred embodiment, the presentation structure is a coiled-coilstructure, allowing the presentation of the flourette on an exteriorloop. (See, for example, Myszka et al., Biochem. 33:2362-2373 (1994),hereby incorporated by reference). Using this system investigators haveisolated peptides capable of high affinity interaction with theappropriate target. In general, coiled-coil structures allow for between6 to 20 amino acids.

A preferred coiled-coil presentation structure is as follows:

MGCAALESEVSALESEVASLE SEVAALX_((n)) LAAVKSKL SAVKSKLASVKSKLAACGPP (SEQID NO:49). The underlined regions represent a coiled-coil leucine zipperregion defined previously (see Martin et al., EMBO J. 13(22):5303-5309(1994), incorporated by reference). The bolded X_((n)) region representsthe loop structure and when appropriately replaced with peptides (i.e.fluorettes, generally depicted herein as (X)_(n), where X is an aminoacid residue and n is an integer of at least 5 or 6) can be of variablelength. The replacement of the bolded region is facilitated by encodingrestriction endonuclease sites in the underlined regions, which allowsthe direct incorporation of oligonucleotides at these positions. Forexample, a preferred embodiment generates an XhoI site at the doubleunderlined LE site and a HindIII site at the double-underlined KL site.

In a preferred embodiment, the presentation structure is a minibodystructure. A “minibody” is essentially composed of a minimal antibodycomplementarity region. The minibody presentation structure generallyprovides two randomizing regions that in the folded protein arepresented along a single face of the tertiary structure. (See forexample Bianchi et al., J. Mol. Biol. 236(2):649-59 (1994), andreferences cited therein, all of which are incorporated by reference).Investigators have shown this minimal domain is stable in solution andhave used phage selection systems in combinatorial libraries to selectminibodies with peptide regions exhibiting high affinity, Kd=10⁻⁷, forthe pro-inflammatory cytokine IL-6.

A preferred minibody presentation structure is as follows:MGRNSQATSGFTFSHFYMEWVRGGEYIAASRHKHNKYTTEYSASVKGRYIVSRDTSQSILYLQK KKGPP(SEQ ID NO:50). The bold, underline regions are the regions which may becontain fluoregte sequences. The italicized phenylalanine must beinvariant in the first randomizing region. The entire peptide is clonedin a three-oligonucleotide variation of the coiled-coil embodiment, thusallowing two different randomizing regions to be incorporatedsimultaneously. This embodiment utilizes non-palindromic BstXI sites onthe termini.

In a preferred embodiment, the presentation structure is a sequence thatcontains generally two cysteine residues, such that a disulfide bond maybe formed, resulting in a conformationally constrained sequence. Thisembodiment is particularly preferred when fluorettes are to be secretedfrom a cell. As will be appreciated by those in the art, any number offlourettes, with or without spacer or linking sequences, may be flankedwith cysteine residues. In other embodiments, effective presentationstructures may be generated by the flourettes themselves. For example,the flourettes may be “doped” with cysteine residues which, under theappropriate redox conditions, may result in highly crosslinkedstructured conformations, similar to a presentation structure.Similarly, the flourettes may be controlled to contain a certain numberof residues to confer β-sheet or α-helical structures.

In a preferred embodiment, a presentation structure is neuropeptide headactivator. Bodenmuller et al. EMBO J. 5(8), 1825-1829 (1983) showed thatneuropeptide head activator (HAv) dimerized to yield a biologicallyinactive form of the peptide at concentration as low as 10- ¹³M, thusindicating extremely high self-binding affinity. Aldwin et al. U.S. Pat.No. 5,491,074 observed that a fragment containing the last six aminoacids of this peptide's carboxyl terminus (SKVILF) (SEQ ID NO:51)resulted in dimers that were even more stable than the HA itself. TheU.S. Pat. No. 5,491,074 shows that the last amino acid of SKVILF, the F(phenylalanine), must be on the carboxyl terminus for a proper peptidedimerization activity. However, if F is not on the carboxyl terminus, itmust be followed by one of two amino acids with free carboxyl group, Eor D, to maintain the peptide dimerization. Therefore, if the dimerizingpeptide positioned inside a carrier protein, 6-mer SKVILF sequence mustbe converted to 7-mer SKVILFE (SEQ ID NO:52) or SKVILFD (SEQ ID NO:53)sequences. For example, a presentation structure comprising neuropeptidehead activator peptides comprises: SKVILFE/D(X)_(n)SKVILFE/D (SEQ IDNO:112); wherein X represents any amino acid, and n is at least about 8amino acids. Linkers or spacers as known in the art are optionallyplaced between the HAv peptides and the variable ((X)_(n)) region andoptionally placed at the amino and carboxy terminus of the structure.

Positioning of peptides of between two SKVILFE(D) sequences makes thepeptides constrained. Upon SKVILFE(D)-SKVILFE(D) intramolecularinteraction, the peptide structure is converted from linear to a loop ormini-domain such that the peptide becomes structurally constrained. Thisprovides three important advantages. First, peptides in constrainedconformations usually have higher affinity for a ligand than the samepeptide in linear conformation. Second, the constrained peptide has ahigher protease resistance than the linear peptide as a result of theformation of a “rigid surface” structure. Third, and more importantly,transfer of a constrained peptide as a cassette to a protein carrierwill not dramatically change its original conformation (and consequentlyits binding activity) while a linear peptide is more likely to show adecrease in binding activity.

In a preferred embodiment, the fusion partner is a targeting sequence.As will be appreciated by those in the art, the localization of proteinswithin a cell is a simple method for increasing effective concentration.The localization of a peptide to a specific cellular location, such as amembrane, limits its search for its ligand to that limited dimensionalspace. Alternatively, the concentration of a protein or polypeptide canalso be simply increased by nature of the localization. For example,shuttling the fluorettes into the nucleus confines them to a smallerspace thereby increasing the concentration. Finally, the ligand ortarget may simply be localized to a specific compartment. As will beappreciated by those in the art, when a fluorette is fused to anotherprotein, as outlined herein, the targeting sequence can be fused to theprotein partner as well.

Thus, suitable targeting sequences include, but are not limited to,binding sequences capable of causing binding of the fluorette to apredetermined molecule or class of molecules while retaining activity ofthe fluorette, (for example by using enzyme inhibitor or substratesequences to target a class of relevant enzymes); sequences signallingselective degradation, of itself or co-bound proteins; and signalsequences capable of constitutively localizing the fluorette to apredetermined cellular locale, including a) subcellular locations suchas the Golgi, endoplasmic reticulum, nucleus, nucleoli, nuclearmembrane, mitochondria, chloroplast, secretory vesicles, lysosome, andcellular membrane; and b) extracellular locations via a secretorysignal. Particularly preferred is localization to either subcellularlocations or to the outside of the cell via secretion.

In a preferred embodiment, the targeting sequence is a nuclearlocalization signal (NLS). NLSs are generally short, positively charged(basic) domains that serve to direct the entire protein in which theyoccur to the cell's nucleus. Numerous NLS amino acid sequences have beenreported including single basic NLS's such as that of the SV40 (monkeyvirus) large T Antigen (Pro Lys Lys Lys Arg Lys Val) (SEQ ID NO:54),Kalderon (1984), et al., Cell, 39:499-509; the human retinoic acidreceptor-β nuclear localization signal (ARRRRP) (SEQ ID NO:55); NFκB p50(EEVQRKRQKL SEQ ID NO:56); Ghosh et al., Cell 62:1019 (1990); NFκB p65(EEKRKRTYE (SEQ ID NO:57; Nolan et al., Cell 64:961 (1991); and others(see for example Boulikas, J. Cell. Biochem. 55(1):32-58 (1994), herebyincorporated by reference) and double basic NLS's exemplified by that ofthe Xenopus (African clawed toad) protein, nucleoplasmin (Ala Val LysArg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys Leu Asp)(SEQ ID NO:58), Dingwall, et al., Cell, 30:449-458, 1982 and Dingwall,et al., J. Cell Biol., 107:641-849; 1988). Numerous localization studieshave demonstrated that NLSs incorporated into peptides or grafted ontoproteins not normally targeted to the cell nucleus cause these peptidesand proteins to be concentrated in the nucleus. See, for example,Dingwall, and Laskey, Ann, Rev. Cell Biol., 2:367-390, 1986; Bonnerot,et al., Proc. Natl. Acad. Sci. USA, 84:6795 -6799, 1987; Galileo, etal., Proc. Natl. Acad. Sci. USA, 87:458-462, 1990.

In a preferred embodiment, the targeting sequence is a membraneanchoring signal sequence. This is particularly useful since manyparasites and pathogens bind to the membrane, in addition to the factthat many intracellular events originate at the plasma membrane. Thus,membrane-bound fluorettes are useful for both the identification ofimportant elements in these processes as well as for the discovery ofeffective inhibitors. The invention provides methods for presenting thefluorettes extracellularly or in the cytoplasmic space. Forextracellular presentation, a membrane anchoring region is provided atthe carboxyl terminus of the peptide presentation structure. In anotherembodiment, for extracellular presentation, a membrane anchoring regionis provided at the amino terminus of the peptide presentation structure.Preferably, the amino terminal anchoring region functions as theanchoring region of a Type 2 glycoprotein. The fluorette is expressed onthe cell surface and presented to the extracellular space, such that itcan bind its target fluorophore. The binding of a fluorette, espesciallywhen fused to a polypeptide or protein could confer a function on thecells expressing the fluorette. The cytoplasmic region could be neutralor could contain a domain that, when the fluorette is bound, confers afunction on the cells (activation of a kinase, phosphatase, binding ofother cellular components to effect function). Similarly, the fluorettecould be contained within a cytoplasmic region, and the transmembraneregion and extracellular region remain constant or have a definedfunction.

Membrane-anchoring sequences are well known in the art and are based onthe geometry of mammalian transmembrane molecules. Peptides are insertedinto the membrane based on a signal sequence (designated herein as ssTM)and require a hydrophobic transmembrane domain (herein TM). Thetransmembrane proteins are inserted into the membrane such that theregions encoded 5′ of the transmembrane domain are extracellular and thesequences 3′ become intracellular. Of course, if these transmembranedomains are placed 5′ of the variable region, they will serve to anchorit as an intracellular domain, which may be desirable in someembodiments. ssTMs and TMs are known for a wide variety of membranebound proteins, and these sequences may be used accordingly, either aspairs from a particular protein or with each component being taken froma different protein, or alternatively, the sequences may be synthetic,and derived entirely from consensus as artificial delivery domains.

As will be appreciated by those in the art, membrane-anchoringsequences, including both ssTM and TM, are known for a wide variety ofproteins and any of these may be used. Particularly preferredmembrane-anchoring sequences include, but are not limited to, thosederived from CD8, ICAM-2, IL-8R, CD4 and LFA-1.

Useful sequences include sequences from: 1) class I integral membraneproteins such as IL-2 receptor beta-chain (residues 1-26 are the signalsequence, 241-265 are the transmembrane. residues; see Hatakeyama etal., Science 244:551 (1989) and von Heijne et al, Eur. J. Biochem.174:671 (1988)) and insulin receptor beta chain (residues 1-27 are thesignal, 957-959 are the transmembrane domain and 960-1382 are thecytoplasmic domain; see Hatakeyama, supra, and Ebina et al., Cell 40:747(1985)); 2) class II integral membrane proteins such as neutralendopeptidase (residues 29-51 are the transmembrane domain, 2-28 are thecytoplasmic domain; see Malfroy et al., Biochem. Biophys. Res. Commun.144:59 (1987)); 3) type III proteins such as human cytochrome P450 NF25(Hatakeyama, supra); and 4) type IV proteins such as humanP-glycoprotein (Hatakeyama, supra). Particularly preferred are CD8 andICAM-2. For example, the signal sequences from CD8 and ICAM-2 lie at theextreme 5′ end of the transcript. These consist of the amino acids 1-32in the case of CD8 (MASPLTRFLSLNLLLLGESILGSGEAKPQAP; (SEQ ID NO:59)Nakauchi et al., PNAS USA 82:5126 (1985) and 1-21 in the case of ICAM-2(MSSFGYRTLTVALFTLICCPG (SEQ ID NO:60); Staunton et al., Nature (London)339:61 (1989)). These leader sequences deliver the fluorette to themembrane while the hydrophobic transmembrane domains, placed 3′ of thefluorette region, serve to anchor the construct in the membrane. Thesetransmembrane domains are encompassed by amino acids 145-195 from CD8(PQRPEDCRPRGSVKGTGLDFACDIYIWAPLAGICVALLLSLIITLICYHSR (SEQ ID NO:61);Nakauchi, supra) and 224-256 from ICAM-2(MVIIVTVVSVLLSLFVTSVLLCFIFGQHLRQQR (SEQ ID NO:62); Staunton, supra).

Alternatively, membrane anchoring sequences include the GPI anchor,which results in a covalent bond between the molecule and the lipidbilayer via a glycosyl-phosphatidylinositol bond for example in DAF(PNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT (SEQ ID NO:63), with the boldedserine the site of the anchor; see Homans et al., Nature333(6170):269-72 (1988), and Moran et al., J. Biol. Chem. 266:1250(1991)). In order to do this, the GPI sequence from Thy-1 can becassetted 3′ of the fluorette in place of a transmembrane sequence.

Similarly, myristylation sequences can serve as membrane anchoringsequences. It is known that the myristylation of c-src recruits it tothe plasma membrane. This is a simple and effective method of membranelocalization, given that the first 14 amino acids of the protein aresolely responsible for this function: MGSSKSKPKDPSQR (SEQ ID NO:64) (seeCross et al., Mol. Cell. Biol. 4(9):1834 (1984); Spencer et al., Science262:1019-1024 (1993), both of which are hereby incorporated byreference). This motif has already been shown to be effective in thelocalization of reporter genes and can be used to anchor the zeta chainof the TCR. This motif is placed 5′ of the fluorette in order tolocalize it to the plasma membrane. Other modifications such aspalmitoylation can be used to anchor fluorettes in the plasma membrane;for example, palmitoylation sequences from the G protein-coupledreceptor kinase GRK6 sequence (LLQRLFSRQDCCGNCSDSEEELPTRL (SEQ IDNO:65), with the bold cysteines being palmitolyated; Stoffel et al., J.Biol. Chem 269:27791 (1994)); from rhodopsin (KQFRNCMLTSLCCGKNPLGD (SEQID NO:66); Barnstable et al., J. Mol. Neurosci. 5(3):207 (1994)); andthe p21 H-ras 1 protein (LNPPDESGPGCMSCKCVLS (SEQ ID NO:67); Capon etal., Nature 302:33 (1983)).

In a preferred embodiment, the targeting sequence is a lysozomaltargeting sequence, including, for example, a lysosomal degradationsequence such as Lamp-2 (KFERQ (SEQ ID NO:68); Dice, Ann. N.Y. Acad.Sci. 674:58 (1992); or lysosomal membrane sequences from Lamp-1(MLIPIAGFFALAGLVLIVLIAYLIGRKRSHAGYQTI (SEQ ID NO:69), Uthayakumar etal., Cell. Mol. Biol. Res. 41:405 (1995)) or Lamp-2(LVPIAVGAALAGVLILVLLAYFIGLKHHHAGYEQF (SEQ ID NO:70), Konecki et la.,Biochem. Biophys. Res. Comm. 205:1-5 (1994), both of which show thetransmembrane domains in italics and the cytoplasmic targeting signalunderlined).

Alternatively, the targeting sequence may be a mitrochondriallocalization sequence, including mitochondrial matrix sequences (e.g.yeast alcohol dehydrogenase III; MLRTSSLFTRRVQPSLFSRNILRLQST (SEQ IDNO:71); Schatz, Eur. J. Biochem. 165:1-6 (1987)); mitochondrial innermembrane sequences (yeast cytochrome c oxidase subunit IV;MLSLRQSIRFFKPATRTLCSSRYLL (SEQ ID NO:72);. Schatz, supra); mitochondrialintermembrane space sequences (yeast cytochrome c1;MFSMLSKRWAQRTLSKSFYSTATGAASKSGKLTQKLVTAGVAAAGITASTLLYADSLTAEAMTA (SEQ IDNO:73); Schatz, supra) or mitochondrial outer membrane sequences (yeast70 kD outer membrane protein; MKSFITRNKTAILATVAATGTAIGAYYYYNOLQQQQQRGKK(SEQ ID NO:74); Schatz, supra).

The target sequences may also be endoplasmic reticulum sequences,including the sequences from calreticulin (KDEL; Pelham, Royal SocietyLondon Transactions B; 1-10 (1992)) or adenovirus E3/19K protein(LYLSRRSFIDEKKMP (SEQ ID NO:75); Jackson et al., EMBO J. 9:3153 (1990)).

Furthermore, targeting sequences also include peroxisome sequences (forexample, the peroxisome matrix sequence from Luciferase; SKL; Keller etal., PNAS USA 4:3264 (1987)); farnesylation sequences (for example, P21H-ras 1; LNPPDESGPGCMSCKCVLS (SEQ ID NO:76), with the bold cysteinefarnesylated; Capon, supra); geranylgeranylation sequences (for example,protein rab-5A; LTEPTQPTRNQCCSN (SEQ ID NO:77), with the bold cysteinesgeranylgeranylated; Farnsworth, PNAS USA 91:11963 (1994)); ordestruction sequences (cyclin B1; RTALGDIGN (SEQ ID NO:78); Klotzbucheret al., EMBO J. 1:3053 (1996)).

In a preferred embodiment, the targeting sequence is a secretory signalsequence capable of effecting the secretion of the fluorette. There area large number of known secretory signal sequences which are placed 5′to the fluorette sequence and are cleaved from the peptide region toeffect secretion into the extracellular space. Secretory signalsequences and their transferability to unrelated proteins are wellknown, e.g., Silhavy, et al. (1985) Microbiol. Rev. 49, 398-418. This isparticularly useful to generate a fluorette that is expressed at thesurface of a cell. In a preferred approach, a fusion product isconfigured to contain, in series, secretion signal peptide-presentationstructure-flourette-presentation structure. In this manner, target cellsgrown in the vicinity of cells caused to express the fluorettes, arebathed in secreted fluorette. Target cells exhibiting a phenotypicchange in response to the presence of a fluorette, or internalization ofthe fluorette and binding to intracellular targets, are localized by anyof a variety of methods, such as, FRET analysis (Selvin et al. MethodsEnzymol. 246:300-334 (1995)) as described below.

Suitable secretory sequences are known, including signals from IL-2(MYRMQLLSCIALSLALVTNS (SEQ ID NO:79); Villinger et al., J. Immunol.155:3946 (1995)), growth hormone (MATGSRTSLLLAFGLLCLPWLQEGSAFPT (SEQ IDNO:80); Roskam et al., Nucleic Acids Res. 7:30 (1979)); preproinsulin(MALWMRLLPLLALLALWGPDPAAAFVN (SEQ ID NO:81); Bell et al., Nature 284:26(1980)); and influenza HA protein (MKAKLLVLLYAFVAGDQI (SEQ ID NO:82);Sekiwawa et al., PNAS 80:3563)), with cleavage between thenon-underlined-underlined junction. A particularly preferred secretorysignal sequence is the signal leader sequence from the secreted cytokineIL-4, which comprises the first 24 amino acids of IL-4 as follows:MGLTSQLLPPLFFLLACAGNFVHG (SEQ ID NO:83).

In a preferred embodiment, the fusion partner is a rescue sequence. Arescue sequence is a sequence which may be used to purify or isolate thefluorette (or, in some cases, the nucleic acid encoding it). Thus, forexample, peptide rescue sequences include purification sequences such asthe His₆ tag for use with Ni affinity columns and epitope tags fordetection, immunoprecipitation or FACS (fluorescence-activated cellsorting). Suitable epitope tags include myc (for use with thecommercially available 9E10 antibody), the BSP biotinylation targetsequence of the bacterial enzyme BirA, flu tags, lacZ, and GST.

In a preferred embodiment, a fluorette sequence functions as a rescuesequence. The fluorette because it binds a fluorophore dye can be usedto purify or isolate a molecule to which it is fused or attached byaffinity chromatography using fluorophore dye columns. If the fluorettealters the excitation or emission spectrum of the bound fluorophore dye,as described below, this difference can be used to monitor attachmentand/or elution of the fluorette from the affinity column. Alternatively,the fluorette/dye complex can be used in FACS. If desired, a linkerjoining the fluorette to its fusion partner also contains a convenientsite or sites to seperate the fluorette from the fusion partner, suchas, a unique protease recognition sequences. Alternatively, the affinitycolumn can contain antibody reactive with the fluorette.

In a preferred embodiment, the fusion partner is a stability sequence toconfer stability to the fluorette. Thus, for example, fluorettes may bestabilized by the incorporation of glycines after an amino-terminalmethionine (MG or MGG0), for protection of the fluorette toubiquitination as per Varshavsky's N-End Rule, thus conferring longhalf-life in the cytoplasm. Similarly, two prolines at the C-terminusimpart upon peptides resistance to carboxypeptidase action. The presenceof two glycines prior to the prolines impart both flexibility andprevent structure initiating events in the di-proline to be propagatedinto the fluorette structure. Thus, preferred stability sequences are asfollows: MG(X)_(n)GGPP (SEQ ID NO:84), where (X)_(n) is a fluorette ofany amino acid and n is an integer of at least about 8, as outlinedabove for fluorette length.

In a preferred embodiment, the fusion partner is a dimerizationsequence. A dimerization sequence allows the non-covalent association ofone fluorette to another fluorette, with sufficient affinity to remainassociated under normal physiological conditions. The dimers may behomo- or heterodimers.

Dimerization sequences may be a single sequence that self-aggregates, ortwo sequences. That is, a first fluorette with dimerization sequence 1,and a second fluorette with dimerization sequence 2, such that uponintroduction into a cell and expression of the nucleic acids,dimerization sequence 1 associates with dimerization sequence 2 to formnew random peptide structures. In addition, dimerization sequences canbe used as presentation structures. That is, by putting a firstdimerization sequence at one terminus of the fluorette, and a seconddimerization sequence at the other terminus, similar to some otherpresentation structures, a “cyclized” fluorette can be made.

Suitable dimerization sequences will encompass a wide variety ofsequences. Any number of protein—protein interaction sites are known. Inaddition, dimerization sequences may also be elucidated using standardmethods such as the yeast two hybrid system, and traditional biochemicalaffinity binding studies, or even using the present methods.

The fusion partners may be placed anywhere (i.e. N-terminal, C-terminal,internal) in the structure as the biology and activity permits, althoughin general, N- or C-terminal fusions are preferred to fusions internalto the fluorette sequence.

In another embodiment, a fluorette occupies an internal position withinthe fusion partner as the biology and activity permits. This will allowa means to measure access of the fluorette for its fluorophore dye as ameans of determining structure and function of the region in which thefluorette resides.

In a preferred embodiment, the fusion partner includes a linker ortethering sequence. Linker sequences between various targeting sequences(for example, membrane targeting sequences) and the other components ofthe constructs and fluorettes may be desirable to allow the candidateagents to interact with potential targets unhindered. For example,useful linkers include glycine-serine polymers (including, for example,(GS)_(n) (SEQ ID NO:85), (GSGGS)_(n) (SEQ ID NO:86) and (GGGS)_(n) (SEQID NO:87), where n is an integer of at least one), glycine-alaninepolymers, alanine-serine polymers, and other flexible linkers such asthe tether for the shaker potassium channel, and a large variety ofother flexible linkers, as will be appreciated by those in the art.Glycine-serine polymers are preferred since both of these amino acidsare relatively unstructured, and therefore may be able to serve as aneutral tether between components. Secondly, serine is hydrophilic andtherefore able to solubilize what could be a globular glycine chain.Third, similar chains have been shown to be effective in joiningsubunits of recombinant proteins such as single chain antibodies. As ismore fully outlined below, linkers such as these may also be used insome embodiments between fluorettes and the moiety to which they arefused.

In alternative embodiments linkers can also be sequences that arederived from other proteins and have no structure or an assumedstructure. In another embodiment the linkers distance the fluorette froma molecule to which they are attached, for example, a fusion protein.

In addition, the fusion partners, including presentation structures, maybe modified, randomized, and/or matured to alter the presentationorientation of the fluorette. For example, determinants at the base ofthe loop may be modified to slightly modify the internal loop tertiarystructure, which maintains the fluorette amino acid sequence.

In a preferred embodiment, combinations of fusion partners are used.Thus, for example, any number of combinations of presentationstructures, targeting sequences, rescue sequences, and stabilitysequences-may be used, with or without linker sequences.

In a preferred embodiment; the fluorette is fused to a target analyte,for example, to monitor or follow the target analyte, as is more fullydescribed below. By “target analyte” or “analyte” or grammaticalequivalents herein is meant any molecule, compound or particle to bedetected. As will be appreciated by those in the art, a large number ofanalytes may be detected using the present methods; basically, anytarget analyte to which a fluorette may be attached may be detectedusing the methods of the invention.

Suitable analytes include organic and inorganic molecules, includingbiomolecules. In a preferred embodiment, the analyte may be anenvironmental pollutant (including pesticides, insecticides, toxins,etc.); a chemical (including solvents, polymers, organic materials,etc.); therapeutic molecules (including therapeutic and abused drugs,antibiotics, etc.); biomolecules (including hormones; cytokines,proteins, lipids, carbohydrates, cellular membrane antigens andreceptors (neural, hormonal, nutrient, and cell surface receptors) ortheir ligands, etc); whole cells (including procaryotic (such aspathogenic bacteria) and eukaryotic cells, including mammalian tumorcells); viruses (including retroviruses, herpesviruses, adenoviruses,lentiviruses, etc.); and spores; etc. Particularly preferred analytesare proteins.

In a preferred embodiment, the target analyte is a protein and thefluorette is made as a fusion protein, using techniques well known inthe art. For example, the nucleic acid encoding the fluorette is linkedin-frame with a nucleic acid encoding a target protein to produce afusion nucleic acid. Preferably, fusion proteins are produced byculturing a host cell transformed with an expression vector containingthe fusion nucleic acid, under the appropriate conditions to induce orcause expression of the fusion protein. The conditions appropriate forfusion protein expression will vary with the choice of the expressionvector and the host cell, and will be easily ascertained by one skilledin the art through routine experimentation. For example, the use ofconstitutive promoters in the expression vector will require optimizingthe growth and proliferation of the host cell, while the use of aninducible promoter requires the appropriate growth conditions forinduction. In addition, in some embodiments, the timing of expression isimportant.

Alternatively, fluorettes can be produced by in vitro transcription ofthe encoding nucleic acid and translation of the RNA transcript, asknown in the art.

In a preferred embodiment, the target analyte is other than a protein,and the fusion of the fluorette to the target analyte is generally donechemically. In general, the fluorette and the target analyte areattached through the use of functional groups on each that can then beused for attachment. Preferred functional groups for attachment areamino groups, carboxy groups, oxo groups and thiol groups. Thesefunctional groups can then be attached, either directly or indirectlythrough the use of a linker. Linkers are well known in the art; forexample, homo-or hetero-bifunctional linkers as are well known (see 1994Pierce Chemical Company catalog, technical section on cross-linkers,pages 155-200, incorporated herein by reference). In an additionalembodiment, carboxyl groups (either from the surface or from thecandidate agent) may be derivatized using well known linkers (see thePierce catalog). For example, carbodiimides activate carboxyl groups forattack by good nucleophiles such as amines (see Torchilin et al.,Critical Rev. Therapeutic Drug Carrier Systems, 7(4):275-308 (1991),expressly incorporated herein). Proteinaceous species may also beattached using other techniques known in the art, for example for theattachment of antibodies to polymers; see Slinkin et al., Bioconj. Chem.2:342-348 (1991); Torchilin et al., supra; Trubetskoy et al., Bioconj.Chem. 3:323-327 (1992); King et al., Cancer Res. 54:6176-6185 (1994);and Wilbur et al., Bioconjugate Chem. 5:220-235 (1994), all of which arehereby expressly incorporated by reference). It should be understoodthat the fluorettes and target analytes may be attached in a variety ofways, including those listed above. What is important is that manner ofattachment does not significantly alter the functionality of thefluorette (and preferably the target analyte as well); that is, theattachment should be done in such a flexible manner as to allow theinteraction of the fluorette peptide with a dye, and the target analyteto its binding partners.

In a preferred embodiment the fluorette will bind to Texas Red. Suitablefluorettes in this embodiment include, but are not limited to,KHVQYWTQMFYS (SEQ ID NO:1); DFLQWKLARQKP (SEQ ID NO:2); KPVQYWTQMFYT(SEQ ID NO:15); KPAQYWTQMFYS (SEQ ID NO:16); KNVQYWTQMFYT (SEQ IDNO:17); KHVQYWTHMFYT (SEQ ID NO:18); KHVQYWTQMFYT (SEQ ID NO:19);NHVHYWTQMFYS (SEQ ID NO:20); THVQYWTQMFYS (SEQ ID NO:21); RTIWEPKEASNHT(SEQ ID NO:105): WSKMGHTVT (SEQ ID NO:106); RWTWEPISE (SEQ ID NO:107);GNQKCLQHNRCST (SEQ ID NO:108); SQTWSFPEH (SEQ ID NO:109); EPMARPWERKQDR(SEQ ID NO:110); and GTLSATRPYGRQW (SEQ ID NO:111).

In a preferred embodiment the fluorette will bind to Rhodamine. Suitablefluorettes in this embodiment include, but are not limited to,IPHPPMYWTRVF (SEQ ID NO:3); IPHRPMYWTPVF (SEQ ID NO:22); andLPHPPMYWTRVF (SEQ ID NO:23).

In a preferred embodiment the fluorette will bind to Oregon Green 514.Suitable fluorettes in this embodiment include, but are not limited to,HGWDYYWDWTAW (SEQ ID NO:4); ASDYWDWEWYYS (SEQ ID NO:5); YPNDFEWWEYYF(SEQ ID NO:6); HTSHISWPPWYF (SEQ ID NO:7); LEPRWGFGWWLK (SEQ ID NO:8);QYYGWYYDHNFW (SEQ ID NO:9); YMYDEYQYWNFW (SEQ ID NO: 10); HEWEYYWDWTAW(SEQ ID NO:24); HEWDYYWDWTAW (SEQ ID NO:25); HGWDYYWDWTDW (SEQ IDNO:26); HGWDYYWDWTPW (SEQ ID NO:27); HGWDYYWDWTTW (SEQ ID NO:28);HGWDYNWDWTAW (SEQ ID NO:29); and QGWDYYWDWTAW (SEQ ID NO:30).

In a preferred embodiment the fluorette will bind to Fluorescein.Suitable fluorettes in this embodiment include, but are not limited to,YPNDFEWWEYYF (SEQ ID NO:6); ASDYWDWEWYYS (SEQ ID NO:5); WYDDWNDWHAWP(SEQ ID NO:11); WHMSPSWGWGYW (SEQ ID NO:12); HMSWWEFYLVPP (SEQ IDNO:13); YWDYSWMHYYAPT (SEQ ID NO:14); YPNEFDWWDYYY (SEQ ID NO:31);YPNDFEWWDYYY (SEQ ID NO:32); YHNDYEWWEYYY (SEQ ID NO:33); YPNDFEWWEYYY(SEQ ID NO:34); YPNDFDWWEYYL (SEQ ID NO:35); YTHDYEWWEYYF (SEQ IDNO:36); YPNDYEWWEYYF (SEQ ID NO:37); YPSDFEWWEYYF (SEQ ID NO:38);YHDFEWWEYYF (SEQ ID NO:39); and YPYDFEWWEYYM (SEQ ID NO:40).

Included within the definition of fluorettes are derivative or variantfluorettes. Accordingly, as used herein, a peptide is a fluorette if theoverall homology of the peptide sequence to the amino acid sequencesshown above (SED ID NOS: 1-40 and 104-110) is preferably greater thanabout 70%, more preferably greater than about 75% even more preferablygreater than about 80% and most preferably greater than 90%, withhomologies of greater than 95 to 98% being especially preferred.Homology in this context means sequence similarity or identity, withidentity being preferred.

This homology will be determined using standard techniques known in theart, including, but not limited to, the local homology algorithm ofSmith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biool. 48:443 (1970),by the search for similarity method of Pearson & Lipman, PNAS USA85:2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Drive, Madison, Wis.), theBest Fit sequence program described by Devereux et al., Nucl. Acid Res.12:387-395 (1984), preferably using the default settings, or byinspection. Preferably, percent identity is calculated by FastDB basedupon the following parameters: mismatch penalty of 1; gap penalty of 1;gap size penalty of 0.33; and joining penalty of 30, “Current Methods inSequence Comparison and Analysis,” Macromolecule Sequencing andSynthesis, Selected Methods and Applications, pp 127-149 (1988), Alan R.Liss, Inc.

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments. It can also plot a tree showing the clusteringrelationships used to create the alignment. PILEUP uses a simplificationof the progressive alignment method of Feng & Doolittle, J. Mol. Evol.35:351-360 (1987); the method is similar to that described by Higgins &Sharp CABIOS 5:151-153 (1989). Useful PILEUP parameters including adefault gap weight of 3.00, a default gap length weight of 0.10, andweighted end gaps.

Another example of a useful algorithm is the BLAST algorithm, describedin Altschul et al., J. Mol. Biol. 215, 403-410, (1990) and Karlin etal., PNAS USA 90:5873-5787 (1993). A particularly useful BLAST programis the WU-BLAST-2 program which was obtained from Altschul et al.,Methods in Enzymology, 266: 460-480 (1996);http://blast.wustl/edu/blast/ README.html]. WU-BLAST-2 uses severalsearch parameters, most of which are set to the default values. Theadjustable parameters are set with the following values: overlap span=1,overlap fraction=0.125, word threshold (T)=11. The HSP S and HSP S2parameters are dynamic values and are established by the program itselfdepending upon the composition of the particular sequence andcomposition of the particular database against which the sequence ofinterest is being searched; however, the values may be adjusted toincrease sensitivity. A % amino acid sequence identity value isdetermined by the number of matching identical residues divided by thetotal number of residues of the “shorter” sequence in the alignedregion. The “longer” sequence is the one having the most actual residuesin the aligned region (gaps introduced by WU-Blast-2 to maximize thealignment score are ignored).

An additional useful algorithm is gapped BLAST as reported by Altschulet al. Nucleic Acids Res. 25:3389-3402. Gapped BLAST uses BLOSUM-62substitution scores; threshold T parameter set to 9; the two-hit methodto trigger ungapped extensions; charges gap lengths of k a cost of 10+k;X_(u) set to 16, and X_(g) set to 40 for database search stage and to 67for the output stage of the algorithms. Gapped alignments are triggeredby a score corresponding to ˜22 bits.

A percent amino acid sequence identity value is determined by the numberof matching identical residues divided by the total number of residuesof the “longer” sequence in the aligned region. The “longer” sequence isthe one having the most actual residues in the aligned region (gapsintroduced by WU-Blast-2 to maximize the alignment score are ignored).

Alternatively, percent sequence identity is determined by inspection inwhich only identities are scored positively (+1) and all forms ofsequence variation including gaps are assigned a value of “0”. Percentsequence identity can be calculated, for example, by dividing the numberof matching identical residues by the total number of residues of the“shorter” sequence in the aligned region and multiplying by 100. The“longer” sequence is the one having the most actual residues in thealigned region.

Once identified, the amino acid sequences of the fluorettes may bemodified as needed. Sequence modifications include substitutions,deletions, insertions or any combination thereof may be used to arriveat a final derivative. Generally these changes are done on a few aminoacids to minimize the alteration of the molecule. However, largerchanges may be tolerated in certain circumstances. When smallalterations in the characteristics of the fluorette are desired,substitutions are generally made in accordance with the following chart:

Original Residue Exemplary Substitutions Ala Ser Arg Lys Asn Gln, HisAsp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn, Gln Ile Leu, Val LeuIle, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met, Leu, Tyr Ser Thr ThrSer Trp Tyr Tyr Trp, Phe Val Ile, Leu

Substantial changes in function are made by selecting substitutions thatare less conservative than those shown in Chart 1, although thesegenerally are not preferred. For example, substitutions may be madewhich more significantly affect the structure of the fluorette backbonein the area of the alteration, for example the alpha-helical orbeta-sheet structure; the charge or hydrophobicity of the molecule; orthe bulk of the side chain. The substitutions which in general areexpected to produce the greatest changes in the fluorette's propertiesare those in which (a) a hydrophilic residue, e.g. seryl or threonyl, issubstituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl,phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substitutedfor (or by) any other residue; (c) a residue having an electropositiveside chain, e.g. lysyl, arginyl, or histidyl, is substituted for (or by)an electronegative residue, e.g. glutamyl or aspartyl; or (d) a residuehaving a bulky side chain, e.g. phenylalanine, is substituted for (orby) one not having a side chain, e.g. glycine.

In a preferred embodiment, not more than about three substitutions ordeletions will be made, and that the change will not be more than about20 number %, usually not more than about 10 number %, of the number ofamino acids in the fluorette, although in some instances higher numbersof alterations may be made.

Similarly, if the function or affinity of the fluorette is to bedecreased, the amount of changes may also be greater. Preferred areconservative substitutions, as known in the art, including substitutionswithin the large hydrophobic group: isoleucine, leucine, valine andphenylalanine; between serine and threonine; glycine and alanine;asparagine and glutamine; aspartic acid and glutamic acid; or lysine,arginine and histidine. In some embodiments, non-conservativealterations are done.

As will be appreciated by those skilled in the art, a number of methodsexist for the creation, isolation, or selection of fluorettes from alibrary of sequences (Oldenburg et al., Proc. Natl. Acad. Sci. U.S.A.89:5393-5397 (1992); Scott et al., Proc. Natl. Acad. Sci. U.S.A.89:5398-5402 (1992); Cull et al., Proc. Natl. Acad. Sci. U.S.A.89:1865-1869 (1992); Devlin et al. Science 249:404-406 (1990); Schatz.Biotechnology 11:1138-1143 (1993)). In preferred embodiments, suchlibraries are based on, for example, either bacteriophage (Matthews andWells, Science 260:1113-1117 (1993); Koivunen et al. Biotechnology13:265-270 (1995); Yu and Smith. Methods Enzymol. 267:3-27 (1996);Harrison et al. Methods Enzymol. 267:83-109 (1996); Rebar and Pabo.Science 263:671-673 (1994); Rebar et al., Methods Enzymol. 267:129-149(1996); Saggio and Laufer. Biochem. J. 293:613-616 (1993); Katz.Biochemistry 34:15421-15429 (1995); Caparon et al. Mol. Divers.1:241-246 (1996); Parmley and Smith. Gene 73:305-318 (1988)), forexample M13 or plasmids (Cull et al. Proc. Natl. Acad. Sci. U.S.A.89:1865-1869 (1992); Schatz et al. Methods Enzymol. 267:171-191 (1996);) or polysomes (Mattheakis et al. Proc. Natl. Acad. Sci. U.S.A.91:9022-9026; Hanes and Pluckthun Proc. Natl. Acad. Sci. U.S.A. 94:4937-4942; Roberts and Szostak. Proc. Natl. Acad. Sci. U.S.A.94:12297-12301)) Alternatively, peptide libraries can be expressed onthe surface of a eukaryotic or prokaryotic cell and directly bound to afluorophore dye and sorted, for example, by FACS. Alternatively, thecell expressing a fluorette is bound to a fluorophore dye that is boundto bead or a magnetic bead and purified and amplified as known in theart.

In a preferred embodiment, fluorettes are created, isolated, or selectedfrom aptamer library phage display approaches. Such libraries are basedon, for example, M13 filamentous bacteriophage (Smith.Science.228:1315-1317 (1985); Cwirla et al. Proc. Natl. Acad. Sci. U.S.A.87:6378-6382 (1990); Devlin et al. Science 249:404-406 (1990); Scott andSmith. Science 249:386-390 (1990)) In a preferred embodiment, fluorettesare identified by screening a phage display peptide library thatcontains a combinatorial library of peptides interposed betwen theleader sequence and the amino terminus of mature form of bacteriophageM13amp9 minor coat protein, pIII.

The recombinatorial library can be produced by a number of methods asknown in the art. In a preferred embodiment, the library is produced byannealing and cloning an oligonucleotide library of sequences into theM13amp9 pIII gene. Annealing of the oligonucleotide library to producedouble-stranded DNA preferably produces “sticky ends” to facilitatedirectional cloning into the appropriate reading frame of the M13amp9.Alternatively, the annealed oligonucleotide library contains uniquerestriction enzyme sites, which are digested to produce “sticky ends” tofacilitate directional cloning. The oligonucleotide library preferablyalso encodes an in-frame linker, tethering sequence, or spacer asdescribed herein. The function of the linker is to decrease or minimizesteric hindrance and promote accessibility of the fluorette for itsfluorophore dye. The linker also promotes structural and functionalindependence of the fluorette. Phage particles bearing the peptidelibrary are produced by introducing the M13amp9 vector into a permissivebacterial host, such as, Escherichia coli and other gram negativebacteria. According to the above strategy for cloning the peptidelibrary, during phage maturation, the leader secretory sequence of pIIIis removed, resulting in the peptide library sequence positionedimmediately at the amino-terminus of the mature protein.

Cloning and expression of the oligonucleotide library produces a libraryof peptides either directly fused to the pIII protein or, preferably,fused via a linker or spacer. The linker or spacer can be, for example,sequence, as described herein, interposed between the fluorette and thepIII protein sequences.

The nucleic acids which give rise to the fluorettes are chemicallysynthesized, and thus may incorporate any nucleotide at any position.Thus, when the nucleic acids are expressed to form peptides, any aminoacid residue may be incorporated at any position. The synthetic processcan be designed to generate randomized nucleic acids, to allow theformation of all or most of the possible combinations over the length ofthe nucleic acid, thus forming a library of randomized nucleic acids andpeptides. In an alternative embodiment, random DNA fragments may be madefrom fragmentation of genomic DNA, followed by sizing, and ligation intoan appropriate vector for expression and selection.

The library should provide a sufficiently structurally diversepopulation of randomized expression products to effect aprobabilistically sufficient range to allow isolation of a fluorette.Accordingly, a library must be large enough so that at least one of itsmembers will have a structure that gives it affinity for a fluorophoredye. Although it is difficult to gauge the required absolute size of alibrary, nature provides a hint with the immune response: a diversity of10⁷-10⁸ different antibodies provides at least one combination withsufficient affinity to interact with most potential antigens faced by anorganism. Published in vitro selection techniques have also shown that alibrary size of 10⁷ to 10⁸ is sufficient to find structures withaffinity for the target. A library of all combinations of a peptide fromabout 7 to 20 amino acids in length has the potential to code for20⁷(10⁹) to 20²⁰. Thus, with libraries of 10⁷ to 10⁸ per ml of phageparticles the present methods allow a “working” subset of atheoretically complete interaction library for 7 amino acids, and asubset of shapes for the 20²⁰ library. Thus, in a preferred embodiment,at least 10⁶, preferably at least 10⁸, more preferably at least 10¹⁰ andmost preferably at least 10¹² different expression products aresimultaneously analyzed in the subject methods. Preferred methodsmaximize library size and diversity.

It is important to understand that in any library system encoded byoligonucleotide synthesis one cannot have complete control over thecodons that will eventually be incorporated into the peptide structure.This is especially true in the case of codons encoding stop signals(TAA, TGA, TAG). In a synthesis with NNN as the random region, there isa 3/64, or 4.69%, chance that the codon will be a stop codon. Thus, in apeptide of 10 residues, there is an unacceptable high likelihood that46.7% of the peptides will prematurely terminate. For free peptidestructures this is perhaps not a problem. But for larger structures,such as those envisioned here, such termination will lead to sterilepeptide expression. To alleviate this, random residues are encoded asNNK, where K=T or G. This allows for encoding of all potential aminoacids (changing their relative representation slightly), but importantlypreventing the encoding of two stop residues TAA and TGA. Thus,libraries encoding a 10 amino acid peptide will have a 15.6% chance toterminate prematurely. For candidate nucleic acids which are notdesigned to result in peptide expression products, this is notnecessary. In one embodiment, the library is fully randomized, with nosequence preferences or constants at any position.

Once generated a peptide library is screened for binding to afluorescent dye. In a preferred embodiment, bacteriophage expressingfluorettes are selected by biopanning, which consists of four sequentialsteps: i) phage binding with a fluorophore dye carrier, ii) removal ofunbound or weakly bound phage, iii) elution of bound phage and (iv)amplification of bound phage. Certain steps can be omitted, for example,such as washing and amplification if the peptide library is expressed ineukaryotic or mammalian cells and fluorescence is detected and cells areshorted according to fluorescence or spectral shifts. If necessary ordesirable, the amplified phage are used for the subsequent round(s) ofbiopanning against the corresponding fluorophore dye carrier untilapparent enrichment for binding is observed over background. Preferably,after about four rounds of biopanning, phage bearing sequences that bindspecifically to a fluorophore dye can be identified. Followingbiopanning, the individual phage are isolated, amplified and thesequence of the fluorette can be determined by various methods as knownin the art. In another embodiment, eukaryotic or prokaryotic cellsexpressing a peptide library on their surface can be sorted by FACSand/or magnetic beads to isolate cells expressing fluorettes.

For biopanning, the fluorophore dyes can be bound to solid-phasecarriers. In a preferred embodiment, the dyes are covalently attached toa target bead. This facilitates washing and removal of non-specificallybound or weakly bound phage. Preferably, a linker or spacer molecule isinterposed between the fluorophore dye and the target bead, for example,succinimidy esters and derivatives of fluorophores. A spacer moleculeincreases the accessibility of the fluorophore dye and minimizespotential steric hindrance that may interfere with the interactionsbetween the fluorophore dye and the bacteriophage particles that bearthe peptide library. The number of fluorophore dye molecules bound tothe carrier will vary. In general, there are up to about 1 micromole.Higher or lower amounts of dye can be attached per ml of carrier beads,as needed, for example to increase or decrease the stringency of thebiopanning conditions.

Once a fluorette is identified, it can be mutagenized or derivatized toisolate fluorettes with altered properties. In a preferred embodiment,the derivatize fluorettes have a higher affinity or specificity,thereby, providing, for example, increased sensitivity for the assay orsystem in which the fluorette is employed. This also allows the use oflower amounts of fluorescent dye, thereby, limiting toxicity of thefluorescent dye to cells, if needed.

In a preferred embodiment, affinity maturation (Yu and Smith. supra) canbe used to produce mutant or derivative fluorettes with improvedaffinities over their corresponding parental fluorettes. To create,select or isolate fluorettes with a higher affinity, a secondoligonucleotide library is produced that is, preferabbly, based. Thatis, some positions within the sequence are either held constant, or areselected from a limited number of possibilities. For example, in apreferred embodiment, the nucleotides or amino acid residues arerandomized within a defined class, for example, of hydrophobic aminoacids, hydrophilic residues, sterically biased (either small or large)residues, towards the creation of cysteines, for cross-linking, prolinesfor SH-3 domains, serines, threonines, tyrosines or histidines forphosphorylation sites, etc., or to purines, etc.

In a preferred embodiment, the amino acid sequences of the fluorettesare subjected to biased mutagenesis at a desired mutagenesis rate usinga number of methods as known in the art. The sequences can bemutagenized by systemmatic alteration of the codons encoding thefluorettes. Oligonucleotide mutagenesis, is preferably, performed duringthe synthesis of the oligonucleotide library. To increase theprobability that all amino acids will be represented at each positionand to limit the generation of stop codons within the fluorette encodingsequences, specific nucleotides, such as A and C, can be omitted fromthe third position of each codon. As described above, theoligonucleotide library is preferably annealed and cloned into anexpression vector of choice.

To select fluorettes with higher binding affinity than the parentalfluorette, the biopanning conditions are preferably altered to be morestringent by, for example, changing one or more of the followingconditions. This also provides a method to maximize selection againstthe parental fluorette. To increase the stringency of the biopanning,the concentration of the fluorophore dye bound to the carrier beads canbe decreased. In addition, the phage concentration represented in thebinding step and/or the binding time also can be reduced. Alternatively,the stringency of the washing conditions is increased. Washingstringency can be increased by, for example, increasing the volume ofwashing buffer per ml of carrier beads, increasing the wash temperature,altering the ionic strength or pH of the wash buffer, increasing thedetergent concentration or by using a stronger, ionizing detergent.(Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual (2ndEdition). Cold Spring Harabor Laboratory, Cold Spring Harbor, N.Y.).Other parameters that can be altered to increase stringency are, forexample, increasing the temperature and competition for binding to thefluorophore dye by related molecules.

Fluorette binding to a fluorophore dye can be assessed in several ways.For example, when a phage display system is used, binding of thebacteriophage-bearing peptide library to the fluorophore dye-carrier canbe determined by comparing the infectious particle titers of input phageto eluted phage. To measure phage-fluorophore dye binding in solution, aknown concentration of free fluorophore dye is mixed with a known numberof purified phage particles. The number of particles is preferablycalculated from the optical densities of the purifed phage at 260 nm and280 nm but other methods, such as, infectious titer can be used.Phage-fluorophore dye complexes are separated by, for example,precipitation to remove unbound dye, spotted onto a solid-supportfilter, such as, nitrocellulose and scanned for fluorescence. Othermethods to remove unbound dye include, for example, competition withrelated molecules, washing, and increased stringency To determinenonspecific binding, controls using phage particles that do not displayrecombinatorial library sequences are run in parallel. In addition,specific to nonspecific binding ratios also can be quantitated.

Fluorette/fluorophore dye dissociation constants can be determined inseveral ways. For example, when a phage display system is used,phage-fluorophore dye dissociation constants can be determined by, forexample, incubating for several hours subsaturating amounts of phagewith a fluorophore dye bound to a carrier, for example, a bead asdescribed above. The bead suspensions are removed, for example, bycentrifugation, and the unbound phage in the supernatants can betitrated. Other methods for measuring unbound phage are known in theart, for example, visualization. Dissociation constants can be measuredvia a standard linear Scatchard plot. Nonspecific background binding isdetermined using phage that does not express fluorette sequences.

In a preferred embodiment, fluorettes are encoded by nucleic acids andproduced by recombinant molecular biology techniques (Sambrook et al.(1989) Molecular Cloning: A Laboratory Manual (2nd Edition) Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.; Ausubel et al. (1994)Current Protocols in Molecular Biology. Massachusettes General Hospitaland Harvard Medical School. Boston, Mass). Thus, the present inventionprovides nucleic acids encoding fluorettes. By “nucleic acids” herein ismeant a nucleic acid, DNA or RNA depending on the delivery or expressionvehicle or vector used, which can be manipulated to express fluorettes;that is, the nucleic acids encoding the fluorettes and the fusionpartners and linkers if present. In addition, the nucleic acids willalso generally contain enough extra sequence to effect translation ortranscription, as necessary. For a fluorette, the nucleic acid generallycontains cloning sites which are placed to allow in frame expression ofthe fluorettes, and any fusion partners, if present, such aspresentation structures. For example, when presentation structures areused, the presentation structure will generally contain the initatingATG, as a part of the parent vector. For example, for retrovirusexpression, the nucleic acids are generally constructed with an internalCMV promoter, tRNA promoter or cell specific promoter designed forimmediate and appropriate expression of the fluorette at the initiationsite of RNA synthesis. The RNA is expressed anti-sense to the directionof retroviral synthesis and is terminated as known, for example with anorientation specific terminator sequence. Interference from upstreamtranscription is alleviated in the target cell with theself-inactivation deletion, a common feature of certain retroviralexpression systems. In another embodiment, other expression vectors orsystems also can be used, such as, adenovirus, adenoassociated virus, ortransposons.

Generally, the nucleic acids are introduced and expressed within thecells to produce fluorettes. By “introduced into” or grammaticalequivalents herein is meant that the nucleic acids enter the cells in amanner suitable for subsequent expression of the nucleic acid. Themethod of introduction is largely dictated by the targeted cell type,discussed below. Exemplary methods include CaPO₄ precipitation, liposomefusion, lipofectin®, electroporation, viral infection, etc. Thecandidate nucleic acids may stably integrate into the genome of the hostcell (for example, with retroviral introduction), or may exist eithertransiently or stably in the cytoplasm (i.e. through the use oftraditional plasmids, utilizing standard regulatory sequences, selectionmarkers, etc.). As many pharmaceutically important screens require humanor model mammalian cell targets, retroviral vectors capable oftransfecting such targets are preferred.

In a preferred embodiment the fluorettes functions within the cell inwhich they are expressed. Alternatively, fluorettes may be expressedwithin a cell or secreted from a cell, and purified. If desired, thefluorette can be introduced into another cell by electroporation,calcium phosphate precipitation, microinjectioin, liposome fusion,lipofectin, viral infection, etc. Fluorettes also may be produced invitro by transcription/translation reactions. Alternatively, fluorettesare chemically synthesized using any of a number of methods.

In a preferred embodiment, the present invention provides a complex orcomposition of a fluorette bound to a fluorophore dye. The binding ofthe fluorette to the fluorophore dye leads to four possible outcomes inregards to the excitation and emission spectra of the fluorophore dye i)little to no change in the fluorophore emission or excitation spectra,ii) change in fluorophore emission, iii) change in fluorophoreexcitation and (iv) change in both fluorophore emission and excitations.

In one embodiment, binding of the fluorette to the fluorophore dye mayprevent fluorophore excitation or emission. In either scenario, thefluorophore dye, no longer fluoresces when bound to the fluorette. Inanother embodiment, the excitation spectrum and/or the emission spectrumof the fluorophore dye is shifted, to a shorter or longer-wavelength.Alternatively, the excitation spectrum and/or emission spectrum can bebroadened.

In a preferred embodiment the excitation spectrum is altered up to about5 nm, about 10 nm, being preferred, 20 nm particularly preferred, andabout 100 nm being especially preferred.

Fluorettes in their various forms find use in detector systems andvarious types of assays and techniques using a large set of potentialfluorophore dyes, for example as, in detection of viral deliverysystems, diagnostics, high-throughput assays.

Once made, the compositions find use in a number of applications. Theseapplications include binding a fluorophore dye to the fluorette,removing unbound dye and detecting fluorescence. In a preferredembodiment, the emission and/or excitation spectrum of the fluorophoredye is altered when bound to a fluorette. Therefore, it is not necessaryto remove unbound dye. In a preferred embodiment, are applications thatin some manner tethers the excitation and emission of two or moreflourette/fluorescent dye complexes in such a way that they can bedetected by fluorescence resonance energy transfer (FRET).

FRET applications based on fluorette/fluorophore dye interactionsprovide methods to demonstrate the proximity or interactions of two ormore molecules. These molecules include, for example, biologicalmolecules, such as proteins, lipids, nucleic acids, carbohydrates. Inanother embodiment, FRET can be applied to detect or induce thelocalized activation of a produrg

In a preferred embodiment, the interactions of two or more proteins,each containing a unique fluorette sequence can be demonstrated asfollows. First, the fluorophore dyes are bound by their correspondingfluorettes. Next, one of the fluorette/fluorophore dye complexes ordonor complex is excited and fluoresces. When the two proteinscontaining the fluorette sequences are in close proximity or in some wayhave interacted, the emitted light of the first donor complex causes thesecond fluorette/fluorophore dye complex (acceptor) to fluoresce.

Another example of a FRET application employs fluorette/fluorophore dyecomplexes that are covalently or associately linked to targetbiomaterials via small ligands. The demonstration of FRET as describedabove indicates an interaction or the proximity of the targetbiomaterials when bound to their respective ligands. Other applicationsinclude, “trigger dependent FRET”, in which the use and design oftrigger molecules, such as, heavy metal ions, drugs, etc., thatspecifically bind to the ligand or target biomaterials, causingdissociation of one or more of the fluorette/dye complexes from thetarget biomaterials and a decrease or loss of FRET.

FRET may also be employed to detect intramolecular interactions orevents, such as, changes in molecular structure that occurs in amolecule with the binding and/or dissociation of a ligand.

In FRET applications, double or higher order fluorettes may be linked,for example, via a rigid connector, such as polyalanine or polyserine,to provide the correct angle of orientation of corresponding donor andacceptor fluorette/fluorophore dye complexes. This may be of importancefor efficient and quantitative FRET output. In some cases, it may bedesirable for the spacer to contain a peptide sequence or other materialwhich, upon binding a specific fluorophore dye, brings the donor andacceptor comlexes into proximity such that FRET can ensue.

FRET applications also include dual fluorophore dye quenching due tofluorette binding. For example, fluorettes specific for donor andacceptor fluorophores are joined by a protease cleavable spacer. Thisfluorette dimer is bound to its two fluorophore dyes targets and FRETensues as described above. However, FRET decreases after proteasecleavage of the fluorette dimer.

Other novel assays are based on fluorophore dye quenching due to itsbinding of a specific fluorette. For example, a target protein islabeled in vitro by a fluorophore dye and is assembled into a complexparticle or structure with other materials (e.g., other proteins, DNA orRNA) whose conformation or structure is to be investigated. The additionof a specific fluorette after the complex is assembled and measuringfluoresence indicates if the fluorette had access (binding) or no access(no binding) to its target dye.

Novel reporter gene assays based on fluorette-induced Stokes shiftchanges also can be created. For example, a fluorette fused with a givencarrier protein can be used as a gene activity reporter for the presenceof the protein. Cell extracts, or living cells expressing the protein,can be monitored for fluorette activity when the specific fluorophoredye is bound by the peptide. Binding of the fluorophore dye by thefluorette/peptide modifies the excitation and/or emitted wavelengthpermitting specific detection of the expressed protein. Consequently,the more Stokes shift changes introduced by binding of peptide the moresensitive the assay can become. The simplicity of such an approachsurpasses the currently available enzymatic reporter gene assays (e.g.,luciferase and beta-galactosidase assays).

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.All references, patents, patent applications, and publications citedherein are incorporated by reference in their entirety.

EXAMPLE 1 Synthesis of Fluorophore Dye Carriers

Four fluorophore dyes, Texas Red, Fluorescein, Rhodamine Red and OregonGreen 514, were chosen, for selection of fluorettes from a phage displaylibrary (Example 2). The chemical structures of the dye conjugates areshown in FIG. 1. The first three dyes, Texas Red, Fluorescein andRhodamine Red fall into one group: (i) they have a relatively high molarextinction coefficient for absorption and excellent quantum fluorescenceyields (Haugland supra), (ii) they have non-overlapping spectralcharacteristics, (iii) they have the potential for cross-quenchingand/or FRET analysis. The fourth dye, Oregon Green 514, relatedstructurally to Fluorescein, was chosen to determine ifstructure-function relationships could be discerned between fluorettepeptides that bound related dyes.

The extinction coefficient and quantum yield is important forsensitivity of detection and was a major factor determining choice offluorophores. Fluorescence detection by the outlined approach, at itssimplest conception, is stochiometric in nature. In addition, peptidebinding of the target dye might lead to some fluorescence quenching.Finally, if fluorettes can be created that each can specifically binddifferent dyes then it is possible to measure proximal interactionsbetween molecules by FRET relative to the binding of specific dyeshaving accommodating overlapping fluorescence spectra.

Succinimidyl esters of the four chosen fluorophore dyes (with 3- or7-atom spacer between the dye molecule and the reactive succinimidylgroup) were separately and covalently linked to target beads. Linkageswere established through the formation of a stable peptide bond toUltraLink Immobilized DADPA carrier beads (capacity −45 μMoles of freeamino groups per ml of beads) containing a 12-atom diaminodipropylaminespacer and a terminal amino-group (Pierce). Activated derivatives offluorophore dyes: 6-(fluorescein-5-(and-6)-carboxamido)hexanoic acid,succinimidyl ester (5(6)-SFX), “mixed isomers”; Oregon Green 514carboxylic acid, succinimidyl ester; Rhodamine Red-X, succinimidylester, “mixed isomers” and Texas Red-X, succinimidyl. ester, “mixedisomers” were purchased from Molecular Probes.

Covalent coupling of activated derivatives of fluorophore dyes to apolymer carrier was performed in accordance with both manufacturer'sprotocols, with some modifications. Briefly, 1 ml of each dye derivative(5 mg/ml in anhydrous dimethylformamide) was separately mixed with 15 mlof 50% slurry of UltraLink Immobilized DADPA in 0.2M sodium bicarbonatebuffer, pH 8.3 (the coupling yield was at least 95% and, consequently,four fluorophore dye carriers contained 0.7-1 μMoles of covalently bounddyes per ml of beads), unreacted amino groups of the carriers wereextensively acetylated by an addition of 18-fold molar excess of aceticacid N-hydroxysuccinimide ester (Sigma) followed by hydroxylaminetreatment of the fluorophore dye carriers in order to destroy anyunstable intermolecular intermediates of fluorophore dyes via theirhydroxyl groups (for Fluorescein- and Oregon Green 514 carriers only).Finally, fluorophore dye carriers were quenched in 0.75M Tris-HCl, pH8.7, loaded to the columns and extensively washed in high salt buffer,50 mM Tris-HCl, pH 7.5+1M NaCl followed by a storage buffer, 50 mMTris-HCl, pH 7.5+150 mM NaCl (TBS)+0.05% sodium azide: Fluorophore dyecarriers were stored as 50% slurries at 4° C.

The chemical structures of the dye conjugates are shown in FIG. 1. Theresulting total spacer between the dye molecule and polymer carrier was19-atoms long for Texas Red-, Fluorescein-, Rhodamine Red-conjugates and15-atoms long for Oregon Green 514-conjugate. This allows maximalreduction of steric hindrance for potential interactions with the largebacteriophage particles that bear the peptide libraries. Afterpreparation we determined that the fluorophore dye carriers containedfrom 0.7-1 μMoles of covalently bound dye per ml of carrier beads.

EXAMPLE 2 Selection for Peptide Aptamers that Bind Small MoleculeFluorophores

A phage display peptide library was screened that contained acombinatorial library of 12-mer peptides fused via a short glycinelinker spacer (GGG) to the amino-terminus of a minor coat pIII protein(5 copies per particle, Li et al. (1980) J. Biol. Chem. 255:10331-10337)of the filamentous bacteriophage M13mp19 (Ph.D. phage display, NewEngland Biolabs). During phage maturation, the leader secretory sequenceis removed. This results in the 12-mer peptide positioned immediately atthe amino-terminus of the mature protein.

E. coli ER2537 strain [F′ lacI^(q) Δ(lacZ)M15 proA⁺B⁺/fhuA2 supE thiΔ(lac-proAB) Δ(hsdSM-mcrB)5 (rk⁻ mk⁻ mcrBC⁻)] and E. coli TG-1 strain[supE thi-1 Δ(lac-proAB) Δ(mcrB-hsdSM)5 (rk⁻ mk⁻) (F′ traD36 proABlacI^(q)ZΔM15)] were purchased from New England Biolabs and Stratagene,respectively, and used as bacteriophage M13mp19 hosts. Allbacteriological techniques were performed as described (Sambrook et al.,supra; Ausubel et al., supra).

Each biopanning round consisted of four sequential steps: (i) phagebinding with a fluorophore dye carrier, (ii) washing unbound phage fromthe beads; (iii) nonspecific elution of bound phage and (iv)amplification of bound phage. The Ph.D.-12 phage display peptide library(based on modified M13mp19 bacteriophage, 1.9×10⁹ independenttransformants) as a part of the Ph.D.-12 phage display peptide librarykit was purchased from New England Biolabs. All procedures were carriedout at room temperature unless noted. First, 0.15 ml of centrifugedfluorophore dye carrier beads were blocked with 3 ml of TBS+2 mg/ml BSA(blocking buffer) for 1.5 hr with gentle rotating, beads were washedwith 15 ml TBS+0.1% Tween-20+0.5mg/ml BSA (binding buffer), centrifuged,mixed with 1.8×10¹¹ plaque-forming units (pfu) of the Ph.D.-12 phagedisplay peptide library (^(—.)95-fold library size) in 3 ml of bindingbuffer and suspension was gently rotated for 4 hr. For washing, beadsuspensions were centrifuged and beads were transferred to themicrocolumns and slowly washed by 100-fold beads volume of TBS+0.1%Tween-20. The bound phage were nonspecifically eluted by 1 ml of 0.2Mglycine-HCl, pH 2.2+1 mg/ml BSA (elution buffer) for 10′ and the eluateswere immediately adjusted to a neutral pH by 0.15 ml of 1M Tris-HCl, pH9.1. Bound phage yields were determined by a titration of the eluates onthe ER2537 host strain (Sambrook et al., supra). Amplification step: allbound-phage (1.1 ml) were added to 27 ml of 1/100 diluted overnightER2537 culture and amplified for 4.5 hr at 37° C. with vigorous shaking.The amplified phage were precipitated from cell supernatants by ⅕ V of20% PEG+2.5M NaCl at 4° C., re-precipitated again and, finally,suspended in 0.5 ml TBS+0.05% sodium azide. The resultant ⁻50-foldconcentrated amplified phage were stored at 4° C. and their titersusually were in the range 0.5-1.5×10¹³ pfu/ml. For long-term storageamplified phage were adjusted to 50% glycerol and stored at −20° C.

The amplified phage were used for the next biopanning rounds against thecorresponding fluorophore dye carrier, if necessary, until apparentenrichment for binding was observed over background. We observed asignificant increase in the amounts of bound phage after 3-4 rounds ofbiopanning (for all of the fluorophore dye carriers see FIG. 2). Thiswas compared to the nonspecific phage background in the first and secondbiopanning rounds. Eluted phage from round 4 selected against fourdifferent fluorophore dye carriers are termed herein as TR-4, RhR-4,OG-4 and Flu-4 (standing for Texas Red, Rhodamine Red, Oregon Green 514and Fluorescein carriers, respectively).

The amplified phage from round 1 were further selected against thecorresponding fluorophore dye carrier. The next biopanning rounds wereperformed in the same manner as round 1, except that: (i) input phage inthe binding reaction were increased to 4.5×10¹¹ pfu, (ii) time ofbinding was reduced to 2 hr in round 4, (iii) Tween-20 concentration ina washing buffer was increased to 0.2% in round 3 and 0.4% in round 4,(iv) eluted phage from last round 4 were not amplified, but ratherserved as a source of independent phage clones used for sequencing.

The TR-4, RhR-4, OG-4 and Flu-4 phage sets were used as a source ofindependent phage clones that were sequenced for further analysis. Phagesingle-stranded DNAs (ss DNAs) and double-stranded DNAs (ds DNAs) werepurified as previously described (Sambrook et al., supra).Fluorette-coding portions of the DNA and adjacent DNA regions weresequenced with −96 gIII sequencing primer CCCTCATAGTTAGCGTAACG (NewEngland Biolabs) (SEQ ID NO:115) using an Applied Biosystems 391automated DNA sequencer.

The sequences from the insert regions of the phage are grouped inTable 1. Two unique sequences were found that bound to the Texas Redconjugate beads, one for Rhodamine Red, seven for Oregon Green, and sixfor Fluorescein. Note that the Oregon Green set and the Fluorescein setshared two sequences that were identical at the nucleotide level as wellas the amino acid sequence presented. These and other issues relevant tosuch observations are explored later.

Phage selected against the Texas Red carrier gave rise to the sequencesKHVQYWVTQMFYS (SEQ ID NO:1) and DFLQWKLARQKP (SEQ ID NO:2)at a 5:1 ratio(Table 1). Biopanning with the phage display library against theRhodamine Red carrier gave rise to a single phage clone, RhR401,carrying the amino acid sequence IPHPPMYWTRVF. Thus, TR-4 and RhR-4phage sets may be considered to be nearly “pure” phage clone populationsby the fourth round of selection.

We tested the binding specificity and excluded the possibility that theTR401 and RhR401 phage were selected against the polymer linker moietyrather than a fluorophore dye moiety of the dye carriers. The TR-4 andRhR-4 phage were cross-bound to Rhbdamine Red and Texas Red carriers,respectively. Cross-binding of each phage to the inappropriate dyecarrier did not exceed the nonspecific phage background binding (datanot shown). Thus the TR401 and RhR401 fluorettes are specific by thiscomparison for their respective conjugated dyes, despite the similarityof the compound core ring structures. Second, they had affinity for thecorresponding conjugated fluorophore dyes rather than only a polymerlinker moiety of Texas Red and Rhodamine Red carriers or other chemicalfeatures of the carriers themselves.

TABLE 1 Fluorophore dye-specific phage clones and peptide fluorettesequences. Phage Phage clone Fluorette net Fluuorette Fluorophore dyecarrier clone^(a) frequency Fluorette Fluorette consensus^(b) charge^(c)hydrophobicity^(d) Texas Red TR401^(e) 5/6  KHVQYWTQMFYS (SEQ ID NO: 1)unique sequence +1 5/12 TR406 1/6  DFLQWKLARQKP (SEQ ID NO: 2) (with asingle +2 5/12 exception) Rhodamine Red RhR401^(e) 6/6  IPHPPMYWTRVF(SEQ ID NO: 3) unique sequence +1 5/12 Oregon Green 514 OG403^(e) 4/12HGWDYYWDW TAW (SEQ ID NO: 4) YWDW −2 7/12 OG401^(e) 2/12 ASDYWDWEWYYS(SEQ ID NO: 5) W (D/E) YY −3 7/12 OG402^(e) 2/12 YPNDFEWWEYYF (SEQ IDNO: 6) YY −3 7/12 OG409 1/12 HTSHISWPPWYF (SEQ ID NO: 8) NFW   0 5/12OG410 1/12 LEPRWGFGWWLK (SEQ ID NO: 8) +1 6/12 OG411 1/12 QYYGWYYDHNFW(SEQ ID NO: 9) −1 7/12 OG412 1/12 YMYDEYQYWNFW (SEQ ID NO: 10) −2 7/12Fluorescein OG402^(e) 7/14 YPNDFEWWEYYF (SEQ ID NO: 6) YY −3 7/12OG401^(e) 2/14 ASDYWDWEWYYS (SEQ ID NO: 5) −3 7/12 Flu406^(e) 2/14WYDDWNDWHAWP (SEQ ID NO: 11) −3 6/12 Flu404 1/14 WHMSPSWGWGYW (SEQ IDNO: 12)   0 5/12 Flu405 1/14 HMSWWEFYLVPP (SEQ ID NO: 13) −1 6/12 Flu4131/14 YWDYSWHYYAPT (SEQ ID NO: 14) −1 7/12 ^(a)phage clones were isolatedafter four biopanning rounds with random combinatorial Ph.D.-12 phagedisplay peptide library (see text), ^(b)partial consensus (shownunderlined) for Oregon Green 514- and Fluorescein-specific peptidefluorettes, (YWDW (SEQ ID NO: 113); W(D/E)YY (SEQ ID NO: 114) ^(c)−1 (D,E) or +1 (K, R), ^(d)number of hydrophobic amino acids (A, V, L, I, W,Y, F) per total number of peptide fluorette amino acids, ^(e)phage cloneis represented in the corresponding group at least twice.

Four rounds of biopanning with the phage display peptide library againstOregon Green 514 and Fluorescein carriers were undertaken. Three cloneswere predominant in the sequenced population selected against OregonGreen (OG401, OG402, OG403) and four clones were represented once (Table1). Similarly, when selected against Fluorescein three clonespredominated and others were represented only once. Notably two of thethree predominant Fluorescein-specific fluorettes had the same sequenceas two of the predominant, independently selected fluorettes that hadbeen found with the Oregon Green 514 carrier. These were OG402 fluorette(YPNDFEWWEYYF(SEQ ID NO:6)) and the OG401 fluorette (ASDYWDWEWYYS(SEQ IDNO:5)). Since Oregon Green 514 is structurally related to Fluorescein,and is considered a Fluorescein pentafluoride (see FIG. 1), independentselection of the same peptide fluorettes against Oregon Green 514 andFluorescein carriers was predicted. The same fluorettes selectedindependently against these fluorophores possibly bind to similardomains of the Oregon Green 514 and Fluorescein dye molecules.

The selected phage had been originally selected as being capable ofbinding fluorophore dyes that had been covalently linked to a polymercarrier. The nature of carrier-crosslinked fluorophores, while usefulfor initial selection of phage, is inappropriate to study affinity ofthe phage except in relative terms. We therefore tested whether theselected phage can bind to the corresponding free fluorophore dyes insolution. Each bacteriophage particle contains 5 copies ofpIII-fluorette fusion protein. Therefore phage binding is governed byavidity considerations. We bound free fluorophore dyes to highlyconcentrated phage solutions and assayed for bound fluorophore dye afterprecipitation of the phage.

Four activated derivatives of fluorophore dyes (see above) were quenchedin 0.5M Tris-HCl, pH 8.7 and used as “free” dyes. Ten μl of PEG-purifiedand 300-fold concentrated phage TR401, RhR401, OG402, OG403(1.3-1.8×10¹² phage particles; calculated from optical densities of thepurified phage at 260 and 280 nm) in TBS+0.05% sodium azide buffer wereseparately mixed with an equal volume of 20 μM solution of thecorresponding free dyes in the same buffer; mixtures were adjusted byTween-20 to 0.1% and incubated for 3 hr. Phage-fluorophore dye complexeswere precipitated three times by PEG-8000 in order to remove unbound dyeand dissolved in 6.6 μl of TBS+0.05% sodium azide buffer. Finally,phage-fluorophore dye complexes (1 μl of each) were spotted tonitrocellulose filter and filter was scanned on the Storm 840 scanner(Molecular Dynamics) in Blue Fluorescence/Chemifluorescence mode with200 μ pixel resolution. To determine nonspecific binding the normalizedamounts of phage particles of amplified, unselected, and PEG-purifiedPh.D.-12 phage display peptide library were separately incubated withthe corresponding free dyes and treated in the same conditions. Allbinding experiments were accomplished in duplicate. Specific/nonspecificsignal ratios were quantified by densitometry of spot images using“Measure” option of NIH Image 1.59 freeware package.

We found that OG403, OG402, RhR401 and TR401 phage specifically boundtheir respective fluorophore dyes (see FIG. 3). This result shows thatphage selected against covalently bound fluorophore dye were stillcapable of interacting with their cognate free fluorophore dye insolution.

EXAMPLE 3 Forced Evolution of Higher Affinity Fluorophore-bindingPeptides

The total number of possible random 12-mer peptides is equal to20¹²=4.1×10¹⁵. The complexity of the phage display peptide library usedfor biopanning against the fluorophore dye carriers was much smaller,containing only 1.9×10⁹ clones. Thus, the library represents a fractionof all possible 12-mer peptides that could have been searched forbinding. We therefore sought to improve the present fluorettes. We choseto introduce mutations into the fluorette peptide sequences at the DNAlevel in the phage and then select for phage that displayed peptideswith higher affinity (compared to the parent) against the respectivefluorophore dye carrier. We chose the Texas Red- and RhodamineRed-specific clones (TR401 and RhR401, respectively), as well as thepredominant Oregon Green 514- and Fluorescein-specific fluorette clones(OG403 and OG402, respectively, see above), for this forced evolutionbased on initial indications of their affinities (data not shown). Weset up oligonucleotide synthesis of the corresponding fluorette-codedDNA sequences in such a manner that the mutagenesis rate was 9% forevery 36 nucleotide positions of the peptide fluorette. Nucleotides Aand C were omitted from the third position in each codon to improve therelative representation of all amino acids and by limits stop codongeneration.

Four minus-strand oligonucleotides containing degeneratedfluorette-coding sequences were synthesized (Protein & Nucleic AcidsFacility, Stanford University Medical Center),

TR401-91CL:

CTCCCCTTCGGCCGAACCTCCACCAGAATAAAACATCTGCGTCCAATACTGCACATGCTTAGAGTGAGAATAGAAAGGTACCACTCTCCC (SEQ ID NO:88);

RhR401 -91 CL:

CTCCCCTTCGGCCGAACCTCCACCAAACACACGAGTCCAATACATAGGAGGATGCGGAATAGAGTGAGAATAGAAAGGTACCACTCTCCC (SEQ ID NO:89);

OG402-91 CL:

CTCCCCTTCGGCCGAACCTCCACCAGAATAATACTCCCACCACTCAAAATCATTCGGATAAGAGTGAGAATAGAAAGGTACCACTCTCCC (SEQ ID NO:90);

OG403-91 CL:

CTCCCCTTCGGCCGAACCTCCACCCCAAGCAGTCCAATCCCAATAATAATCCCACCCATGAGAGTGAGAATAGAAAGGTACCACTCTCCC (SEQ ID NO:91).

Nucleotides in a regular case shows no degeneracy; boldfaced nucleotidedesignates 91% of shown nucleotide and 3×3% of each of the other threenucleotides; boldfaced and underlined nucleotide designates 91% of A orC and 9% of C or A, respectively.

Degenerated minus-strand oligonucleotides were separately annealed tothe plus-strand oligonucleotide GGGAGAGTGGTACCTTTCTATTCTCAC (SEQ IDNO:92), partial duplexes were filled by T4 DNA Polymerase (Ausubel etal., supra), cut by KpnI and EagI and, finally, 1.9 ng of doubledigested filled duplexes was ligated to 200 ng of low-melting pointagarose gel-purified large fragment of the replicative form (RF)Ph.D.-121 DNA cut with the same pair of enzymes (the Ph.D.-121 was arandomly picked phage clone from the Ph.D.-12 phage display peptidelibrary) in a total volume 0.1 ml. Ligations were electroporated into0.5 ml of TG-1 electrocompetent cells (Stratagene) according tomanufacturer's protocol and phage library complexities were determinedby immediate mixing of several 10-fold dilutions of transfected TG-1cells with ER2537 cells and plating. Phage libraries were furtheramplified in 400 ml of liquid LB media for 4 hr at 37° C. with vigorousshaking, concentrated by PEG (see above) and, finally, suspended in 0.5ml TBS +0.05% sodium azide buffer.

Titers of four resultant nonrandom combinatorial phage libraries were0.2-4.00 ×10¹³ pfu/ml. For a long-term storage phage libraries wereadjusted to 50% glycerol and stored at −20° C. Twelve independent phageclones from each TR401-91CL and RhR401-91CL libraries were sequenced inorder to determine the average level of amino acid substitutions in thefluorette moiety.

We determined the nucleotide sequences of twelve independent phageclones from each of the TR401-91CL and RhR401-91CL libraries in order todetermine the average level of amino acid substitutions in the displayedfluorettes. These were determined to be 2.30 and 2.45, respectively.These experimental values correlated well to the expected theoreticalvalues (calculated from a degeneration frequency 0.09 on the nucleotidelevel, see above). The nucleotide mutation rate from the other twolibraries, OG403-91CL and OG402-91CL was not determined but likely had acomparable average level of amino acid substitutions in the fluorettemoiety as all four corresponding oligonucleotides were synthesized inparallel using the same batch of pre-mixed monomer nucleotides.

A key difference of the secondary biopanning versus the primarybiopanning with the original phage library is the strict need tomaximize selection against the originating, parental fluorette. For thispurpose we significantly reduced concentrations of the fluorophore dyecarrier. We also reduced the phage concentrations present during thebinding steps. In addition, the binding time was reduced while theamounts of washing buffer per ml of carrier beads were increased.Specifically, (i) input phage in the binding reaction were decreased to1.0×10¹⁰ pfu, (ii) time of binding was reduced to 10′, (iii) amount offluorophore dye carrier beads in the binding reaction was reduced to0.03 ml, (iv) following binding to the phage beads were washed with330-fold beads volume of washing buffer and (v) Tween-20 concentrationin the washing buffer was increased to 0.4%.

An increase in approximately 1.2-1.5 logs of magnitude in the totalnumber of bound phage after 2-3 rounds of biopanning (compared to thatof the first round) for all the fluorophore dye carriers was seen (seeFIG. 4). Eluted phage from round 3 were termed TRS-3, RhRS-3, OGS-3 andFluS-3. These phage represent a second generation of phage clonesselected against the four fluorophore dyes, Texas Red, Rhodamine Red,Oregon Green 514 and Fluorescein, respectively.

We isolated and sequenced a panel of independent phage clones from eachof the TRS-3, RhRS-3, OGS-3 and FluS-3 phage sets to determine thesequences of the mutants (Table 2). In the Texas Red selection half ofthose sequenced encoded the parental amino acid structure. Thisindicated successful, though partial, selection against the parentalbinding characteristics under these-conditions. Four of the sixteenTRS401 progeny cloned carried a conservative S12T substitution. Each ofthese substitutions occurred independently as it can be observed theyare associated with additional, independent mutations. This suggeststhat the S12T substitution is probably an important change that enhancesthe affinity of the phage for the dye. Three progeny Texas Redfluorettes showed substitution at the second amino acid of histidine toP (found twice) or N. It is interesting to note that clone TRS311carried both the H2P and the S12T mutations. Fewer mutant clones, onlytwo, were observed for Rhodamine Red. The RhR401 phage clone mightrepresent a local optima in affinity for Rhodamine Red. We foundnumerous in-frame deletions in the RhR401 fluorette-coded DNA sequences(about 33% of independent clones carrying large and small in-framedeletions in the fluorette moiety). This is an indicator of potentialtoxicity of the substituted mutant RhR401 fluorette sequences forbacteriophage growth and survival (data not shown). For Oregon Green 514multiple different peptide sequences were selected, with only twoindependently containing the same, non-conservative, mutation (G2E). ForFluorescein the majority of peptide fluorettes selected were also notparental sequences. Several independent mutations were independentlyselected at fluorette positions 3 (N to S found twice), 5 (conservativechange of F to Y found three times), 6 (conservative change E to D foundtwice), 9 (conservative E to D found twice), and 12 (conservative F to Yfound 4 times). Also, certain

TABLE 2 Fluorophore dye-specific phage clones and peptide fluorettesequences. The second generation. Number of amino Phage clone acidsubstitutions Fluorophore dye carrier Phage clone^(a) frequency in thefluorette Fluorette^(b) Texas Red TR401 parent^(c) 8/16 — KHVQYWTQMFYS(SEQ ID NO: 1) TRS311 1/16 2 .P.........T TRS310 1/16 2 .PA.........TRS315 1/16 2 .N.........T TRS313 1/16 2 .......H...T TRS305 2/16 1...........T ...........T TRS304 1/16 2 N..H........ TR5308 1/16 1T........... Rhodamine Red RhR401 parent^(c) 13/15  — IPHPPMYWTRVF (SEQID NO: 3) RhRS308 1/15 2 ...R.....P.. RhRS307 1/15 1 L........... OregonGreen 514 OG403 parent^(c) 4/16 — HGWDYYWDWTAW (SEQ ID NO: 4) OGS3161/16 2 .E.E........ OGS312 1/16 1 .E.......... OGS303 5/16 1..........D. ..........D. ..........D. ..........D. OGS308 2/16 1..........P. ..........P. OGS305 1/16 1 ..........T. OGS302 1/16 1.....N...... OGS301 1/16 1 Q........... Fluorescein OG402 parent^(c)5/16 — YPNDFEWWEYYF (SEQ ID NO: 6) FluS303 1/16 4 ...E.D..D..Y FluS3021/16 2 ........D..Y FluS315 1/16 3 .H..Y......Y FluS310 1/16 1...........Y FluS307 1/16 2 .....D.....L FluS311 1/16 3 .TH.Y.......FluS312 1/16 1 ....Y....... FluS313 1/16 1 ..S......... FluS316 1/16 1..S......... FluS304 1/16 1 .-H......... FluS314 1/16 2 ..Y........M^(a)phage clones were isolated after three biopanning rounds withTR401-91CL, RhR401-91CL, OG403-91CL and OG402-91CL nonrandomcombinatorial phage display peptide libraries (see text), ^(b)amino acidsubstitutions in mutant peptide fluorette vs the corresponding parentare shown, mutant peptide fluorette sequence is shown as many times asit was found (see phage clone frequency); dots designate the same aminoacid as that in the corresponding parent; dash in a second position ofthe FluS304 fluorette designates in-frame deletion, ^(c)originalparental clones and the parental clones carrying silent mutation(s) inpeptide fluorette moiety.

positions appeared favored for change, i.e., 2, 3, and 12 with no strongbias for the substituted residue.

These mutations may be important for increasing the overall affinity ofthe peptide fluorette-fluorophore interaction. We tested one of each ofthe phage from the secondary screens for increases in avidity (Table 3).PEG-purified TR401 phage, TRS311 phage or the amplified Ph.D.-12 phagedisplay peptide library phage as a negative control (input1.5×10⁸-1.1×10¹⁰ pfu with 2 to 5-fold increments) were incubated with23.5 μl of BSA-blocked and washed Texas Red carrier beads in bindingbuffer (see above) in total volume 40 μl for 3 hr. Beads suspensionswere centrifuged, the supernatants (unbound phage) were titrated anddissociation constants were measured via a standard linear Scatchardplot. For TR401 and TRS311 phage bound/unbound ratios were quitereliable (6.3-13.1 in the range of phage concentrations shown above).All binding experiments were performed in duplicate and titrations wereperformed in triplicate. This direct binding assay can reliably measureK_(d) if it does not exceed ⁻1.5-2 nM. For higher K_(d), bound/unboundratios became unreliable (=1).

Other PEG-purified phage (input 5×10⁸-2.5×10¹⁰ pfu with 2 to 5-foldincrements) were bound to the respective fluorophore dye carriersessentially in the same manner as described above for Texas Red-specificphage. RhR401 and RhRS308, OG403 and OGS316, OG402 and FluS303 phage,were bound to the Rhodamine Red, Oregon Green 514 or Fluoresceincarriers, respectively. Following binding the beads were quickly washedtwice by 0.25 ml of TBS+0.1% Tween-20 and suspended in 10 ml TBS. 10 μlof suspension was mixed with ⁻2×10⁸ log-phase ER2537 cells and incubated1 hr at 4° C. with slight shaking to allow phage adsorption. Severalten-fold dilutions of infected cells were mixed with noninfected ER2537cells and plated in standard plaque assay (Sambrook et al., supra). Allbinding experiments and titrations were accomplished in duplicate.Nonspecific background of binding (determined with amplified Ph.D.-12phage display peptide library phage) did not exceed more than 3% ofspecific binding in each case. For all above phage pfu/particle ratiowas in the range 0.4-0.5.

The Texas Red progeny phage TRS311, which contained the apparentlyimportant S12T substitution, had a threefold increased avidity ascompared to the TR401 parent. The affinity of the double mutant cloneRhRS308 versus the parent was not even marginally improved (Table 3).For Oregon Green 514 the phage clone OGS316, which carried the G2Esubstitution, had a 2.7-fold higher avidity than its parent. Themultiply substituted clone FluS303, which contained two putativelyimportant changes, had a 6.5 fold increase in relative avidity. Thus,peptides can be matured to higher avidity. This likely is due tocorresponding affinity increases for the individual peptide fluoretteagainst its respective dye.

EXAMPLE 4 Binding of Peptides to Fluorophores in Solution

To identify peptide fluorettes that bind fluorophore dye independent ofthe full context of the pIII fusion bacteriophage protein we usedsynthetic peptides corresponding to the sequence of the selectedfluorette region. We added to the peptide eight amino acids derived fromthe adjacent sequence of the pIII protein. This would accommodate anypartial contributions of context from the pIII protein sequence. Thesewere followed by a short GGG spacer and His₆ tag (see Table 4). Wesynthesized peptides from the original library screening that had beencapable in solution of specifically binding each of the three dyes(Texas Red, Oregon Green 514, Fluorescein). The Texas Red peptideschosen were TR401 and its higher avidity progeny TRS311; a peptidecorresponding to the unrelated Texas Red primary clone TR406 was alsosynthesized. The Oregon Green 514 clones OG401 and OG403 weresynthesized to test, in part, the observation that both contained acommon motif YWDW which might represent a common binding motif forOregon Green 514. Note that the OG401 clone also bound to Fluorescein inthe original screening. The other clone which bound to both Oregon Greenand Fluorescein in the primary screen, OG402, was synthesized. Thepeptide derived from a secondary screen against Fluorescein, FluS303,which demonstrated a 6.5-fold higher affinity was also synthesized. Nopeptides for Rhodamine Red were synthesized.

Peptides were bound to cobalt ion-coated Sepharose beads and theresultant peptide-coated beads were incubated with correspondingfluorophore dyes, washed, and visualized for dye binding. The sameamounts of Texas Red-specific peptides PepTR401, PepTRS311, PepTR406 aswell as nonspecific peptide PepControl (see Table 4) were bound via His₆tag to the TALON Metal Affinity Resin (Clontech) in TBS bufferessentially as described by manufacturer's protocol. Beads (5 μl) werewashed twice with 0.5 ml TBS to remove unbound peptide and incubatedwith 0.5 μM Texas Red in 40 μl of TBS buffer for 1 hr at RT. Finally,the beads were washed three times with 0.3 ml TBS to remove unbound dye.Fluorescent and nonfluorescent control beads were photographed with10-fold magnification on fluorescent microscope Axiophot (Zeiss) usingRhodamine Red/Texas Red filter with 5″ exposure.

Most peptides failed to bind dyes to any detectable degree. However,Texas Red peptides PepTR401 and PepTRS311 showed significant binding toTexas Red while they bound to neither Oregon Green 514 nor Fluorescein(data not shown). PepTR406 did not bind to Texas Red, however. Lack ofdye binding might be due to low peptide affinity for cognate dyes and/orbecause the binding requires contributions from the pIII fusion proteinnot present in these peptides.

The above test only checks for bound dye that remains fluorescent at thewavelengths tested. Binding of fluorophore dyes to specific peptidesmight result in changes in the fluorescence spectra of bound dye versusthe spectra of free dye. Or, one might observe an increase or quenchingof fluorescence. We mixed concentrated peptide with 50 nM of thecorresponding dye in solution and determined the excitation and emissionspectra of the resulting mixture. No detectable changes in peakexcitation nor emission were observed for peptides selected againstOregon Green 514 or Fluorescein (data not shown), nor for PepTR406, norfor a control peptide (FIG. 5B).

TABLE 3 Phage - fluorophore dye binding affinity. Dissociationconstants. Affinity increase Fluorophore dye carrier Phage CloneFluorette^(a) K_(d) (nM) (fold)^(b) Texas Red TR401 parent KHVQYWTQMFYS(SEQ ID NO: 1) 0.27 TRS311 .P.........T 0.09 3.0 Rhodamine Red RhR401parent IPHPPMYWTRVF (SEQ ID NO: 3) 23.0 RhRS308 ...R.....P.. 21.5 noincrease Oregon Green 514 OG403 parent HGWDYYWDWTAW (SEQ ID NO: 4) 6.4OGS316 .E.E........ 2.4 2.7 Fluorescein OG402 parent YPNDFEWWEYYF (SEQID NO: 6) 17.4 FluS303 ...E.D..D..Y 2.7 6.5 ^(a)amino acid substitutionsin mutant peptide fluorette vs the corresponding parent are shown; dotsdesignate the same amino acid as that in the corresponding parent;^(b)mutant peptide fluorette vs the corresponding parental peptidefluorette.

However, the peak positions of the excitation and emission spectra ofsoluble Texas Red bound to 10 μM PepTR401 was shifted relative to TexasRed alone or as compared to a mixture of Texas Red and a nonspecificcontrol peptide (FIG. 5A). A +1.9 nm peak excitation shift with PepTR401was observed. The peptide was checked against Fluorescein as anonspecific control and showed no shift in the Fluorescein excitation oremission spectra. An approximate peak excitation shift of +2.8 nm waseffected upon dye binding with PepTRS311. Interestingly, althoughPepTR401 had a shift in its peak excitation, no emission shift changewas elicited upon binding fluorophore. However, PepTRS311 did manifest asignificant +1.4 nM peak emission shift in its spectra. We checked allother excitation and emission wavelengths and the changes observedcorrespond to a largely global shift in the spectra. No othersignificant changes in local optima were observed (data not shown).

Interestingly, the TR401 peptide is the progenitor of the TRS311peptide. Thus, the two substitutions H2P and S12T result not only in anincreased binding, but also in an apparent differences in the excitationspectra elicited. Moreover, though both show a shift in the excitationonly the selected, higher affinity, PepTRS311 had a peak emissionspectra shift. Thus, one or both of the two substitutions, H2P or S12T,are critical for shifting the spectra of the emission profile.

We attempted with these findings to obtain an affinity measurement forthe higher affinity TRS311 peptide. Peptides in different concentrations(1.3-20 μM) were incubated with Texas Red (50 nM) in 0.6 ml of TBSbuffer for 1 hr at RT. Excitation and emission spectra of samples wereobtained by using spectrofluorimeter SPEX Fluoromax (Jobin Yvon-SPEXInstruments Co.) and DataMax software package. Peak positions wereautomatically calculated by software. List mode data was saved for otheranalyses.

Reduction of the concentration of PepTRS311 from 10 μM to 1.3 μM reducedslightly, but not completely, the change in peak excitation position(FIG. 5A). The narrow dynamic range of the shift and the broad emissionspectra provided for a limited ability to obtain an approximate measureof the affinity. Affinity measurements calculated roughly from theseresults place the affinity of the peptide for the fluorophore at 0.1-0.5μM. Further examination of this issue by other approaches is required toobtain more accurate reflections of the affinity and off-rates. However,together with the agarose bead-binding data we can conclude that we havesuccessfully selected for peptides with sufficient affinity to bindsmall molecule fluorophore dyes in solution. In addition, the binding isspecific and the spectral qualities of the dyes can be modulateddependent upon modifications of the bound peptide.

TABLE 4 Fluorette-carrying peptides. Fluorophore dye Peptide^(a)Sequence^(b) Texas Red Pep. TR401 KHVOYWTOMFYS GGGSAETVGGGHHHHHH (SEQ IDNO: 41) Pep. TRS311 KPVOYWTOMFYT GGGSAETVGGGHHHHHH (SEQ ID NO: 42) Pep.TR406 DFLOWKLAROKP GGGSAETVGGGHHHHHH (SEQ ID NO: 43) Oregon Green 514Pep. 0G403 HGWDYYWDWTAW GGGSAETVGGGHHHHHH (SEQ ID NO: 44) Pep. 0G401^(c)ASDYWDWEWYYS GGGSAETVGGGHHHHHH (SEQ ID NO: 45) Pep. OG4O2^(c)YPNDFEWWEYYF GGGSAETVGGGHHHHHH (SEQ ID NO: 46) Fluorescein Pep. FluS303YPNEFDWWDYYY GGGSAETVGGGHHHHHH (SEQ ID NO: 47) Pep. Control^(d)ASGSGASGSAGS GGGSAETVGGGHHHHHH (SEQ ID NO: 48) ^(a)) peptide namesreflect the names of phage clones carrying respective fluorettes exceptPep. Control (see Tables 1 and 2) ^(b)) first twenty amino acids of thepeptides are the same as in the pIII fusion protein of the correspondingphage clones except Pep. Control (see Tables 1 and 2 and also NewEngland Biolabs “Ph.D.-12 Phage Display Peptide Library Kit” manual),thus, amino acids 1-12 are the fluorette potion (double underlined) andamino acids 13-20 are the distal pIII fusion part (underlined). Aminoacids 21-29 contain a small GGG spacer foIlowed by His₆tag (shown inregular case). # (SEQ ID NOS:41-48) ^(c)) specific for both Oregon Green514 and Fluorescein (see Table 1). ^(d)) nonspecific control peptidesynthesized for specificity check.

EXAMPLE 5 Constrained Texas Red-binding Peptides

Constrained Texas Red-binding peptides were identified via five roundsof biopanning of a mixture of two constrained phage display librariesagainst a polymer carrier with covalently bound Texas Red. Two librariescontained SKVILFE-flanked nine or thirteen amino acid variable region inthe N-terminal part of M13 bacteriophage pIII protein. The structure ofthe libraries were as follows:

GGG SKVILFEGPAG (X)_(9 or 13,) GAPG SKVILFEGGPG (SEQ ID NO:93)-(pIIIprotein)

SKVILFE-dimerizers are underlined. Flexible linkers or spacers GGG, GPAG(SEQ.ID NO:94), GAPG (SEQ.ID NO:95) and GGPG (SEQ.ID NO:96) are doubleunderlined. X represents any amino acid.

The flexible linker or spacer GPAG is encoded by a nucleotide sequencecontaining FseI restriction endonuclease site. The flexible linker orspacer GAPG is encoded by a nucleotide sequence containing AscIrestriction endonuclease site. Both FseI and AscI restriction enzymesare rare eight-cutters. Thus, upon a double enzyme digestion, auniversal cassette-library or cassettes containing specific sequencescan be conveniently exchanged between different vector/host systems ordifferent dimerizers of a choice (e.g., pair of cysteines, coiled coilstructures, etc.).

Biopanning against carrier with Texas Red ultimately revealed at leastseven different Texas Red-binding constrained peptides (both 9- and13-mers in a variable part):

GGGSKVILFEGPAG RTIWEPKEASNHT GAPGSKVILFEGGPG (TRP501) SEQ.ID NO:97

GGGSKVILFEGPAG WSKMGHTVT GAPGSKVILFEGGPG (TRP505) SEQ.ID NO:98

GGGSKVILFEGPAG RWTRWEPISE GAPGSKVILFEGGPG (TRP512) SEQ.ID NO:99

GGGSKVILFEGPAG GNQKCLQHNRCST GAPGSKVILFEGGPG (TRP518) SEQ.ID NO:100

GGGSKVILFEGPAG SQTWSFPEH GAPGSKVILFEGGPG (TRP526) SEQ.ID NO:101

GGGSKVILFEGPAG EPMARPWERKQDR GAPGSKVILFEGGPG (TRP527) SEQ.ID NO:102

GGGSKVILFEGPAG GTLSATRPYGRQW GAPGSKVILFEGGPG (TRP541) SEQ.ID NO:103

Consensus motifs can be observed within these peptides (e.g., RXXWEP(SEQ ID NO:104), WEP and TW; see Table 5) that suggests structuralfeatures common to the peptides that allow for efficient binding.Interestingly, no said constrained peptide has a significant homologywith linear Texas Red-binding peptides.

Binding affinity of phage clones revealed the most avid binders, TRP512phage and TRP501 phage, with K_(d) equal to 25 pM and 80 pM,respectively. Other five phage clones were much less avid with K_(d)≦5nM (see Table 5).

Binding of synthetic peptides (nonspecific peptide, TR401, TRP501 andTRP512) to Texas Red is shown in FIG. 6B-C.

Binding affinity of synthetic TRP501 peptide is measured as K_(d)≈200 nM(≈8-fold more avid than linear TR401 and TRS311 peptides). Bindingaffinity of synthetic TRP512 peptide is measured as K_(d)≈10 nM(≈160-fold more avid than linear TR401 and TRS311 peptides).

CONCLUDING REMARKS

The foregoing description details specific methods which can be employedto practice the present invention. Having detailed such specificmethods, those skilled in the art will well enough know how to devisealternative reliable methods at arriving at the same information inusing the fruits of the present invention. Thus, however detailed theforegoing may appear in text, it should not be construed as limiting theoverall scope thereof; rather, the ambit of the present invention is tobe determined only by the lawful construction of the appended claims.All documents cited herein are hereby expressly incorporated byreference.

TABLE 5 Texas Red - specific phage clones carrying SKVILFE - flankedfluorettes. Phage Phage clone Fluorette size Fluorette Fluorette netFluorette Phage clone^(a) frequency (a.a.) Fluorette consensuscharge^(b) hydrophobicity^(c) clone K_(d) TRP501* 42/48  13RTIWEPKEASNHT RXXWEP 0 (−2/+2) 3/13 80 pM (SEQIDNO:105) (SEQIDNO:108)TRP505 1/48 9 WSKMGHTVT WEP +1 2/9 >5 nM (SEQIDNO:106) TRP512 1/48 9RWTWEPISE TW −1 (−2/+1) 3/9 25 pM (SEQIDNO:107) TRP518 1/48 13GNQKCLQHNRCST +2  1/13 >5 nM (SEQIDNO:108) TRP526 1/48 9 SQTWSFPEH −12/9 >5 nM (SEQIDNO:109) TRP527 1/48 13 EPMARPWERKQDR +1 (−3/−4)  2/13 >5nM (SEQIDNO:110) TRP541 1/48 13 GTLSATRPYGRQW +2  4/13 >5 nM(SEQIDNO:111) ^(a)phage clones were isolated after five biopanningrounds with a mixture of combinatorial P71R4-CL-9 & P71R4-Cl-13 phagedisplay peptide libraries ^(b)−1 (D, E) or +1 (K, R) ^(c)number ofhydrophobic amino acids (A, V, L, I, W, Y, F) per total number offluorette amino acids *phage clone is represented in the correspondinggroup at least twice

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 122 <210> SEQ ID NO 1 <211>LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:synthetic <400> SEQUENCE: 1 Lys His Val Gln Tyr Trp Thr Gln Met Phe TyrSer 1 5 10 <210> SEQ ID NO 2 <211> LENGTH: 12 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: synthetic <400> SEQUENCE: 2 Asp PheLeu Gln Trp Lys Leu Ala Arg Gln Lys Pro 1 5 10 <210> SEQ ID NO 3 <211>LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:synthetic <400> SEQUENCE: 3 Ile Pro His Pro Pro Met Tyr Trp Thr Arg ValPhe 1 5 10 <210> SEQ ID NO 4 <211> LENGTH: 12 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: synthetic <400> SEQUENCE: 4 His GlyTrp Asp Tyr Tyr Trp Asp Trp Thr Ala Trp 1 5 10 <210> SEQ ID NO 5 <211>LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:synthetic <400> SEQUENCE: 5 Ala Ser Asp Tyr Trp Asp Trp Glu Trp Tyr TyrSer 1 5 10 <210> SEQ ID NO 6 <211> LENGTH: 12 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: synthetic <400> SEQUENCE: 6 Tyr ProAsn Asp Phe Glu Trp Trp Glu Tyr Tyr Phe 1 5 10 <210> SEQ ID NO 7 <211>LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:synthetic <400> SEQUENCE: 7 His Thr Ser His Ile Ser Trp Pro Pro Trp TyrPhe 1 5 10 <210> SEQ ID NO 8 <211> LENGTH: 12 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: synthetic <400> SEQUENCE: 8 Leu GluPro Arg Trp Gly Phe Gly Trp Trp Leu Lys 1 5 10 <210> SEQ ID NO 9 <211>LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:synthetic <400> SEQUENCE: 9 Gln Tyr Tyr Gly Trp Tyr Tyr Asp His Asn PheTrp 1 5 10 <210> SEQ ID NO 10 <211> LENGTH: 12 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: synthetic <400> SEQUENCE: 10 Tyr MetTyr Asp Glu Tyr Gln Tyr Trp Asn Phe Trp 1 5 10 <210> SEQ ID NO 11 <211>LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:synthetic <400> SEQUENCE: 11 Trp Tyr Asp Asp Trp Asn Asp Trp His Ala TrpPro 1 5 10 <210> SEQ ID NO 12 <211> LENGTH: 12 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: synthetic <400> SEQUENCE: 12 Trp HisMet Ser Pro Ser Trp Gly Trp Gly Tyr Trp 1 5 10 <210> SEQ ID NO 13 <211>LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:synthetic <400> SEQUENCE: 13 His Met Ser Trp Trp Glu Phe Tyr Leu Val ProPro 1 5 10 <210> SEQ ID NO 14 <211> LENGTH: 12 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: synthetic <400> SEQUENCE: 14 Tyr TrpAsp Tyr Ser Trp His Tyr Tyr Ala Pro Thr 1 5 10 <210> SEQ ID NO 15 <211>LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:synthetic <400> SEQUENCE: 15 Lys Pro Val Gln Tyr Trp Thr Gln Met Phe TyrThr 1 5 10 <210> SEQ ID NO 16 <211> LENGTH: 12 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: synthetic <400> SEQUENCE: 16 Lys ProAla Gln Tyr Trp Thr Gln Met Phe Tyr Ser 1 5 10 <210> SEQ ID NO 17 <211>LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:synthetic <400> SEQUENCE: 17 Lys Asn Val Gln Tyr Trp Thr Gln Met Phe TyrThr 1 5 10 <210> SEQ ID NO 18 <211> LENGTH: 12 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: synthetic <400> SEQUENCE: 18 Lys HisVal Gln Tyr Trp Thr His Met Phe Tyr Thr 1 5 10 <210> SEQ ID NO 19 <211>LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:synthetic <400> SEQUENCE: 19 Lys His Val Gln Tyr Trp Thr Gln Met Phe TyrThr 1 5 10 <210> SEQ ID NO 20 <211> LENGTH: 12 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: synthetic <400> SEQUENCE: 20 Asn HisVal His Tyr Trp Thr Gln Met Phe Tyr Ser 1 5 10 <210> SEQ ID NO 21 <211>LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:synthetic <400> SEQUENCE: 21 Thr His Val Gln Tyr Trp Thr Gln Met Phe TyrSer 1 5 10 <210> SEQ ID NO 22 <211> LENGTH: 12 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: synthetic <400> SEQUENCE: 22 Ile ProHis Arg Pro Met Tyr Trp Thr Pro Val Phe 1 5 10 <210> SEQ ID NO 23 <211>LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:synthetic <400> SEQUENCE: 23 Leu Pro His Pro Pro Met Tyr Trp Thr Arg ValPhe 1 5 10 <210> SEQ ID NO 24 <211> LENGTH: 12 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: synthetic <400> SEQUENCE: 24 His GluTrp Glu Tyr Tyr Trp Asp Trp Thr Ala Trp 1 5 10 <210> SEQ ID NO 25 <211>LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:synthetic <400> SEQUENCE: 25 His Glu Trp Asp Tyr Tyr Trp Asp Trp Thr AlaTrp 1 5 10 <210> SEQ ID NO 26 <211> LENGTH: 12 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: synthetic <400> SEQUENCE: 26 His GlyTrp Asp Tyr Tyr Trp Asp Trp Thr Asp Trp 1 5 10 <210> SEQ ID NO 27 <211>LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:synthetic <400> SEQUENCE: 27 His Gly Trp Asp Tyr Tyr Trp Asp Trp Pro ThrTrp 1 5 10 <210> SEQ ID NO 28 <211> LENGTH: 12 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: synthetic <400> SEQUENCE: 28 His GlyTrp Asp Tyr Tyr Trp Asp Trp Thr Thr Trp 1 5 10 <210> SEQ ID NO 29 <211>LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:synthetic <400> SEQUENCE: 29 His Gly Trp Asp Tyr Asn Trp Asp Trp Thr AlaTrp 1 5 10 <210> SEQ ID NO 30 <211> LENGTH: 12 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: synthetic <400> SEQUENCE: 30 Gln GlyTrp Asp Tyr Tyr Trp Asp Trp Thr Ala Trp 1 5 10 <210> SEQ ID NO 31 <211>LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:synthetic <400> SEQUENCE: 31 Tyr Pro Asn Glu Phe Asp Trp Trp Asp Tyr TyrTyr 1 5 10 <210> SEQ ID NO 32 <211> LENGTH: 12 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: synthetic <400> SEQUENCE: 32 Tyr ProAsn Asp Phe Glu Trp Trp Asp Tyr Tyr Tyr 1 5 10 <210> SEQ ID NO 33 <211>LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:synthetic <400> SEQUENCE: 33 Tyr His Asn Asp Tyr Glu Trp Trp Glu Tyr TyrTyr 1 5 10 <210> SEQ ID NO 34 <211> LENGTH: 12 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: synthetic <400> SEQUENCE: 34 Tyr ProAsn Asp Phe Glu Trp Trp Glu Tyr Tyr Tyr 1 5 10 <210> SEQ ID NO 35 <211>LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:synthetic <400> SEQUENCE: 35 Tyr Pro Asn Asp Phe Asp Trp Trp Glu Tyr TyrLeu 1 5 10 <210> SEQ ID NO 36 <211> LENGTH: 12 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: synthetic <400> SEQUENCE: 36 Tyr ThrHis Asp Tyr Glu Trp Trp Glu Tyr Tyr Phe 1 5 10 <210> SEQ ID NO 37 <211>LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:synthetic <400> SEQUENCE: 37 Tyr Pro Asn Asp Tyr Glu Trp Trp Glu Tyr TyrPhe 1 5 10 <210> SEQ ID NO 38 <211> LENGTH: 12 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: synthetic <400> SEQUENCE: 38 Tyr ProAsp Ser Phe Glu Trp Trp Glu Tyr Tyr Phe 1 5 10 <210> SEQ ID NO 39 <211>LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:synthetic <400> SEQUENCE: 39 Tyr His Asp Phe Glu Trp Trp Glu Tyr Tyr Phe1 5 10 <210> SEQ ID NO 40 <211> LENGTH: 12 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: synthetic <400> SEQUENCE: 40 Tyr ProTyr Asp Phe Glu Trp Trp Glu Tyr Tyr Met 1 5 10 <210> SEQ ID NO 41 <211>LENGTH: 29 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:synthetic <400> SEQUENCE: 41 Lys His Val Gln Tyr Trp Thr Gln Met Phe TyrSer Gly Gly Gly Ser 1 5 10 15 Ala Glu Thr Val Gly Gly Gly His His HisHis His His 20 25 <210> SEQ ID NO 42 <211> LENGTH: 29 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: synthetic <400>SEQUENCE: 42 Lys Pro Val Gln Tyr Trp Thr Gln Met Phe Tyr Thr Gly Gly GlySer 1 5 10 15 Ala Glu Thr Val Gly Gly Gly His His His His His His 20 25<210> SEQ ID NO 43 <211> LENGTH: 29 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: synthetic <400> SEQUENCE: 43 Asp Phe Leu Gln TrpLys Leu Ala Arg Gln Lys Pro Gly Gly Gly Ser 1 5 10 15 Ala Glu Thr ValGly Gly Gly His His His His His His 20 25 <210> SEQ ID NO 44 <211>LENGTH: 29 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:synthetic <400> SEQUENCE: 44 His Gly Trp Asp Tyr Tyr Trp Asp Trp Thr AlaTrp Gly Gly Gly Ser 1 5 10 15 Ala Glu Thr Val Gly Gly Gly His His HisHis His His 20 25 <210> SEQ ID NO 45 <211> LENGTH: 29 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: synthetic <400>SEQUENCE: 45 Ala Ser Asp Tyr Trp Asp Trp Glu Trp Tyr Tyr Ser Gly Gly GlySer 1 5 10 15 Ala Glu Thr Val Gly Gly Gly His His His His His His 20 25<210> SEQ ID NO 46 <211> LENGTH: 29 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: synthetic <400> SEQUENCE: 46 Tyr Pro Asn Asp PheGlu Trp Trp Glu Tyr Tyr Phe Gly Gly Gly Ser 1 5 10 15 Ala Glu Thr ValGly Gly Gly His His His His His His 20 25 <210> SEQ ID NO 47 <211>LENGTH: 29 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:synthetic <400> SEQUENCE: 47 Tyr Pro Asn Glu Phe Asp Trp Trp Asp Tyr TyrTyr Gly Gly Gly Ser 1 5 10 15 Ala Glu Thr Val Gly Gly Gly His His HisHis His His 20 25 <210> SEQ ID NO 48 <211> LENGTH: 29 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: synthetic <400>SEQUENCE: 48 Ala Ser Gly Ser Gly Ala Ser Gly Ser Ala Gly Ser Gly Gly GlySer 1 5 10 15 Ala Glu Thr Val Gly Gly Gly His His His His His His 20 25<210> SEQ ID NO 49 <211> LENGTH: 60 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <221> NAME/KEY: UNSURE <222>LOCATION: (28)..(33) <223> OTHER INFORMATION: The x at positions 28through 33 represents any amino acid residue. <223> OTHER INFORMATION:Description of Artificial Sequence: synthetic <400> SEQUENCE: 49 Met GlyCys Ala Ala Leu Glu Ser Glu Val Ser Ala Leu Glu Ser Glu 1 5 10 15 ValAla Ser Leu Glu Ser Glu Val Ala Ala Leu Xaa Xaa Xaa Xaa Xaa 20 25 30 XaaLeu Ala Ala Val Lys Ser Lys Leu Ser Ala Val Lys Ser Leu Ala 35 40 45 SerVal Lys Ser Lys Leu Ala Ala Cys Gly Pro Pro 50 55 60 <210> SEQ ID NO 50<211> LENGTH: 67 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: synthetic <400> SEQUENCE: 50 Met Gly Arg Asn Ser Gln Ala ThrSer Gly Phe Thr Phe Ser His Phe 1 5 10 15 Tyr Met Glu Trp Trp Arg GlyGly Glu Tyr Ile Ala Ala Ser Arg His 20 25 30 Lys His Asn Lys Tyr Thr ThrGlu Tyr Ser Ala Ser Val Lys Gly Arg 35 40 45 Tyr Ile Val Ser Arg Asp ThrSer Gln Ser Ile Leu Tyr Gln Lys Lys 50 55 60 Gly Pro Pro 65 <210> SEQ IDNO 51 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: synthetic <400> SEQUENCE: 51 Ser Lys Val Ile LeuPhe 1 5 <210> SEQ ID NO 52 <211> LENGTH: 7 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: synthetic <400> SEQUENCE: 52 Ser LysVal Ile Leu Phe Glu 1 5 <210> SEQ ID NO 53 <211> LENGTH: 7 <212> TYPE:PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: synthetic <400>SEQUENCE: 53 Ser Lys Val Ile Leu Phe Asp 1 5 <210> SEQ ID NO 54 <211>LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Monkey virus <400> SEQUENCE:54 Pro Lys Lys Lys Arg Lys Val 1 5 <210> SEQ ID NO 55 <211> LENGTH: 6<212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 55 Ala ArgArg Arg Arg Pro 1 5 <210> SEQ ID NO 56 <211> LENGTH: 10 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: synthetic <400>SEQUENCE: 56 Glu Glu Val Gln Arg Lys Arg Gln Lys Leu 1 5 10 <210> SEQ IDNO 57 <211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: synthetic <400> SEQUENCE: 57 Glu Glu Lys Arg LysArg Thr Tyr Glu 1 5 <210> SEQ ID NO 58 <211> LENGTH: 20 <212> TYPE: PRT<213> ORGANISM: Xenopus <400> SEQUENCE: 58 Ala Val Lys Arg Pro Ala AlaThr Lys Lys Ala Gly Gln Ala Lys Lys 1 5 10 15 Lys Lys Leu Asp 20 <210>SEQ ID NO 59 <211> LENGTH: 31 <212> TYPE: PRT <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: synthetic <400> SEQUENCE: 59 Met Ala Ser Pro LeuThr Arg Phe Leu Ser Leu Asn Leu Leu Leu Leu 1 5 10 15 Gly Glu Ser IleLeu Gly Ser Gly Glu Ala Lys Pro Gln Ala Pro 20 25 30 <210> SEQ ID NO 60<211> LENGTH: 21 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: synthetic <400> SEQUENCE: 60 Met Ser Ser Phe Gly Tyr Arg ThrLeu Thr Val Ala Leu Phe Thr Leu 1 5 10 15 Ile Cys Cys Pro Gly 20 <210>SEQ ID NO 61 <211> LENGTH: 51 <212> TYPE: PRT <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: synthetic <400> SEQUENCE: 61 Pro Gln Arg Pro GluAsp Cys Arg Pro Arg Gly Ser Val Lys Gly Thr 1 5 10 15 Gly Leu Asp PheAla Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly 20 25 30 Ile Cys Val AlaLeu Leu Leu Ser Leu Ile Ile Thr Leu Ile Cys Tyr 35 40 45 His Ser Arg 50<210> SEQ ID NO 62 <211> LENGTH: 33 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: synthetic <400> SEQUENCE: 62 Met Val Ile Ile ValThr Val Val Ser Val Leu Leu Ser Leu Phe Val 1 5 10 15 Thr Ser Val LeuLeu Cys Phe Ile Phe Gly Gln His Leu Arg Gln Gln 20 25 30 Arg <210> SEQID NO 63 <211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: synthetic <400> SEQUENCE: 63 Pro Asn Lys Gly SerGly Thr Thr Ser Gly Thr Thr Arg Leu Leu Ser 1 5 10 15 Gly His Cys PheThr Leu Thr Gly Leu Leu Gly Thr Val Thr Met Gly 20 25 30 Leu Leu Thr 35<210> SEQ ID NO 64 <211> LENGTH: 14 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: synthetic <400> SEQUENCE: 64 Met Gly Ser Ser LysSer Lys Pro Lys Asp Pro Ser Gln Arg 1 5 10 <210> SEQ ID NO 65 <211>LENGTH: 26 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:synthetic <400> SEQUENCE: 65 Leu Leu Gln Arg Leu Phe Ser Arg Gln Asp CysCys Gly Asn Cys Ser 1 5 10 15 Asp Ser Glu Glu Glu Leu Pro Thr Arg Leu 2025 <210> SEQ ID NO 66 <211> LENGTH: 20 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: synthetic <400> SEQUENCE: 66 Lys Gln Phe Arg AsnCys Met Leu Thr Ser Leu Cys Cys Gly Lys Asn 1 5 10 15 Pro Leu Gly Asp 20<210> SEQ ID NO 67 <211> LENGTH: 19 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: synthetic <400> SEQUENCE: 67 Leu Asn Pro Pro AspGlu Ser Gly Pro Gly Cys Met Ser Cys Lys Cys 1 5 10 15 Val Leu Ser <210>SEQ ID NO 68 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: synthetic <400> SEQUENCE: 68 Lys Phe Glu Arg Gln 15 <210> SEQ ID NO 69 <211> LENGTH: 36 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: synthetic <400> SEQUENCE: 69 Met Leu Ile Pro IleAla Gly Phe Phe Ala Leu Ala Gly Leu Val Leu 1 5 10 15 Ile Val Leu IleAla Tyr Leu Ile Gly Arg Lys Arg Ser His Ala Gly 20 25 30 Tyr Gln Thr Ile35 <210> SEQ ID NO 70 <211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: synthetic <400> SEQUENCE: 70 Leu Val Pro Ile AlaVal Gly Ala Ala Leu Ala Gly Val Leu Ile Leu 1 5 10 15 Val Leu Leu AlaTyr Phe Ile Gly Leu Lys His His His Ala Gly Tyr 20 25 30 Glu Gln Phe 35<210> SEQ ID NO 71 <211> LENGTH: 26 <212> TYPE: PRT <213> ORGANISM:Yeast <400> SEQUENCE: 71 Met Leu Arg Thr Ser Ser Leu Phe Thr Arg Arg ValGln Pro Ser Leu 1 5 10 15 Phe Ser Arg Asn Ile Leu Arg Gln Ser Thr 20 25<210> SEQ ID NO 72 <211> LENGTH: 25 <212> TYPE: PRT <213> ORGANISM:Yeast <400> SEQUENCE: 72 Met Leu Ser Leu Arg Gln Ser Ile Arg Phe Phe LysPro Ala Thr Arg 1 5 10 15 Thr Leu Cys Ser Ser Arg Tyr Leu Leu 20 25<210> SEQ ID NO 73 <211> LENGTH: 64 <212> TYPE: PRT <213> ORGANISM:Yeast <400> SEQUENCE: 73 Met Phe Ser Met Leu Ser Lys Arg Trp Ala Gln ArgThr Leu Ser Lys 1 5 10 15 Ser Phe Tyr Ser Thr Ala Thr Gly Ala Ala SerLys Ser Gly Lys Leu 20 25 30 Thr Gln Lys Leu Val Thr Ala Gly Val Ala AlaAla Gly Ile Thr Ala 35 40 45 Ser Thr Leu Leu Tyr Ala Asp Ser Leu Thr AlaGlu Ala Met Thr Ala 50 55 60 <210> SEQ ID NO 74 <211> LENGTH: 41 <212>TYPE: PRT <213> ORGANISM: Yeast <400> SEQUENCE: 74 Met Lys Ser Phe IleThr Arg Asn Lys Thr Ala Ile Leu Ala Thr Val 1 5 10 15 Ala Ala Thr GlyThr Ala Ile Gly Ala Tyr Tyr Tyr Tyr Asn Gln Leu 20 25 30 Gln Gln Gln GlnGln Arg Gly Lys Lys 35 40 <210> SEQ ID NO 75 <211> LENGTH: 15 <212>TYPE: PRT <213> ORGANISM: Adenovirus <400> SEQUENCE: 75 Leu Tyr Leu SerArg Arg Ser Phe Ile Asp Glu Lys Lys Met Pro 1 5 10 15 <210> SEQ ID NO 76<211> LENGTH: 19 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: synthetic <400> SEQUENCE: 76 Leu Asn Pro Pro Asp Glu Ser GlyPro Gly Cys Met Ser Cys Lys Cys 1 5 10 15 Val Leu Ser <210> SEQ ID NO 77<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: synthetic <400> SEQUENCE: 77 Leu Thr Glu Pro Thr Gln Pro ThrArg Asn Gln Cys Cys Ser Asn 1 5 10 15 <210> SEQ ID NO 78 <211> LENGTH: 9<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: synthetic <400>SEQUENCE: 78 Arg Thr Ala Leu Gly Asp Ile Gly Asn 1 5 <210> SEQ ID NO 79<211> LENGTH: 20 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: synthetic <400> SEQUENCE: 79 Met Tyr Arg Met Gln Leu Leu SerCys Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser 20 <210> SEQID NO 80 <211> LENGTH: 29 <212> TYPE: PRT <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: synthetic <400> SEQUENCE: 80 Met Ala Thr Gly SerArg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu 1 5 10 15 Cys Leu Pro TrpLeu Gln Glu Gly Ser Ala Phe Pro Thr 20 25 <210> SEQ ID NO 81 <211>LENGTH: 27 <212> TYPE: PRT <213> ORGANISM: Prepoinsulin <400> SEQUENCE:81 Met Ala Leu Trp Met Arg Leu Leu Pro Leu Leu Ala Leu Leu Ala Leu 1 510 15 Trp Gly Pro Asp Pro Ala Ala Ala Phe Val Asn 20 25 <210> SEQ ID NO82 <211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Influenza <400>SEQUENCE: 82 Met Lys Ala Lys Leu Leu Val Leu Leu Tyr Ala Phe Val Ala GlyAsp 1 5 10 15 Gln Ile <210> SEQ ID NO 83 <211> LENGTH: 24 <212> TYPE:PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: synthetic <400>SEQUENCE: 83 Met Gly Leu Thr Ser Gln Leu Leu Pro Pro Leu Phe Phe Leu LeuAla 1 5 10 15 Cys Ala Gly Asn Phe Val His Gly 20 <210> SEQ ID NO 84<211> LENGTH: 14 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <221> NAME/KEY: UNSURE <222> LOCATION: (3)..(10) <223>OTHER INFORMATION: The x at positions 3 through 10 represents afluorotte of any amino acid. <223> OTHER INFORMATION: Description ofArtificial Sequence: synthetic <400> SEQUENCE: 84 Met Gly Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Gly Gly Pro Pro 1 5 10 <210> SEQ ID NO 85 <211>LENGTH: 2 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:synthetic <400> SEQUENCE: 85 Gly Ser 1 <210> SEQ ID NO 86 <211> LENGTH:5 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence: synthetic<400> SEQUENCE: 86 Gly Ser Gly Gly Ser 1 5 <210> SEQ ID NO 87 <211>LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:synthetic <400> SEQUENCE: 87 Gly Gly Gly Ser 1 <210> SEQ ID NO 88 <211>LENGTH: 90 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:synthetic <400> SEQUENCE: 88 ctccccttcg gccgaacctc caccagaata aaacatctgcgtccaatact gcacatgctt 60 agagtgagaa tagaaaggta ccactctccc 90 <210> SEQID NO 89 <211> LENGTH: 90 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: synthetic <400> SEQUENCE: 89 ctccccttcg gccgaacctccaccaaacac acgagtccaa tacataggag gatgcggaat 60 agagtgagaa tagaaaggtaccactctccc 90 <210> SEQ ID NO 90 <211> LENGTH: 90 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: synthetic <400> SEQUENCE: 90 Cys ThrCys Cys Cys Cys Thr Thr Cys Gly Gly Cys Cys Gly Ala Ala 1 5 10 15 CysCys Thr Cys Cys Ala Cys Cys Ala Ala Ala Ala Thr Ala Ala Thr 20 25 30 AlaCys Thr Cys Cys Cys Ala Cys Cys Ala Cys Thr Cys Ala Ala Ala 35 40 45 AlaThr Cys Ala Thr Thr Cys Gly Gly Ala Thr Ala Ala Gly Ala Gly 50 55 60 ThrGly Ala Gly Ala Ala Thr Ala Gly Ala Ala Ala Gly Gly Thr Ala 65 70 75 80Cys Cys Ala Cys Thr Cys Thr Cys Cys Cys 85 90 <210> SEQ ID NO 91 <211>LENGTH: 90 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:synthetic <400> SEQUENCE: 91 ctccccttcg gccgaacctc caccccaagc agtccaatcccaataataat cccacccatg 60 agagtgagaa tagaaaggta ccactctccc 90 <210> SEQID NO 92 <211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: synthetic <400> SEQUENCE: 92 gggagagtgg tacctttctattctcac 27 SEQ ID NO 93 <211> LENGTH: 42 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <221> NAME/KEY: UNSURE <222>LOCATION: (15)..(27) <223> OTHER INFORMATION: The x at positions 15through 27 represents any amino acid. <223> OTHER INFORMATION:Description of Artificial Sequence: synthetic <400> SEQUENCE: 93 Gly GlyGly Ser Lys Val Ile Leu Phe Glu Gly Pro Ala Gly Xaa Xaa 1 5 10 15 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Ala Pro Gly Ser 20 25 30 LysVal Ile Leu Phe Glu Gly Gly Pro Gly 35 40 <210> SEQ ID NO 94 <211>LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:synthetic <400> SEQUENCE: 94 Gly Pro Ala Gly 1 <210> SEQ ID NO 95 <211>LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:synthetic <400> SEQUENCE: 95 Gly Ala Pro Gly 1 <210> SEQ ID NO 96 <211>LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:synthetic <400> SEQUENCE: 96 Gly Gly Pro Gly 1 <210> SEQ ID NO 97 <211>LENGTH: 42 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:synthetic <400> SEQUENCE: 97 Gly Gly Gly Ser Lys Val Ile Leu Phe Glu GlyPro Ala Gly Arg Thr 1 5 10 15 Ile Trp Glu Pro Lys Glu Ala Ser Asn HisThr Gly Ala Pro Gly Ser 20 25 30 Lys Val Ile Leu Phe Glu Gly Gly Pro Gly35 40 <210> SEQ ID NO 98 <211> LENGTH: 38 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: synthetic <400> SEQUENCE: 98 Gly GlyGly Ser Lys Val Ile Leu Phe Glu Gly Pro Ala Gly Trp Ser 1 5 10 15 LysMet Gly His Thr Val Thr Gly Ala Pro Gly Ser Lys Val Ile Leu 20 25 30 PheGlu Gly Gly Pro Gly 35 <210> SEQ ID NO 99 <211> LENGTH: 38 <212> TYPE:PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: synthetic <400>SEQUENCE: 99 Gly Gly Gly Ser Lys Val Ile Leu Phe Glu Gly Pro Ala Gly ArgTrp 1 5 10 15 Thr Trp Glu Pro Ile Ser Glu Gly Ala Pro Gly Ser Lys ValIle Leu 20 25 30 Phe Glu Gly Gly Pro Gly 35 <210> SEQ ID NO 100 <211>LENGTH: 42 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:synthetic <400> SEQUENCE: 100 Gly Gly Gly Ser Lys Val Ile Leu Phe GluGly Pro Ala Gly Gly Asn 1 5 10 15 Gln Lys Cys Leu Gln His Asn Arg CysSer Thr Gly Ala Pro Gly Ser 20 25 30 Lys Val Ile Leu Phe Glu Gly Gly ProGly 35 40 <210> SEQ ID NO 101 <211> LENGTH: 38 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: synthetic <400> SEQUENCE: 101 GlyGly Gly Ser Lys Val Ile Leu Phe Glu Gly Pro Ala Gly Ser Gln 1 5 10 15Thr Trp Ser Phe Pro Glu His Gly Ala Pro Gly Ser Lys Val Ile Leu 20 25 30Phe Glu Gly Gly Pro Gly 35 <210> SEQ ID NO 102 <211> LENGTH: 42 <212>TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: synthetic <400>SEQUENCE: 102 Gly Gly Gly Ser Lys Val Ile Leu Phe Glu Gly Pro Ala GlyGlu Pro 1 5 10 15 Met Ala Arg Pro Trp Glu Arg Lys Gln Asp Arg Gly AlaPro Gly Ser 20 25 30 Lys Val Ile Leu Phe Glu Gly Gly Pro Gly 35 40 <210>SEQ ID NO 103 <211> LENGTH: 42 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: synthetic <400> SEQUENCE: 103 Gly Gly Gly SerLys Val Ile Leu Phe Glu Gly Pro Ala Gly Gly Thr 1 5 10 15 Leu Ser AlaThr Arg Pro Tyr Gly Arg Gln Trp Gly Ala Pro Gly Ser 20 25 30 Lys Val IleLeu Phe Glu Gly Gly Pro Gly 35 40 <210> SEQ ID NO 104 <211> LENGTH: 6<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221>NAME/KEY: UNSURE <222> LOCATION: (2)..(3) <223> OTHER INFORMATION: The xat positions 2 and 3 represents any amino acid. <223> OTHER INFORMATION:Description of Artificial Sequence: synthetic <400> SEQUENCE: 104 ArgXaa Xaa Trp Glu Pro 1 5 <210> SEQ ID NO 105 <211> LENGTH: 13 <212> TYPE:PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: synthetic <400>SEQUENCE: 105 Arg Thr Ile Trp Glu Pro Lys Glu Ala Ser Asn His Thr 1 5 10<210> SEQ ID NO 106 <211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: synthetic <400> SEQUENCE: 106 Trp Ser Lys MetGly His Thr Val Thr 1 5 <210> SEQ ID NO 107 <211> LENGTH: 9 <212> TYPE:PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: synthetic <400>SEQUENCE: 107 Arg Trp Thr Trp Glu Pro Ile Ser Glu 1 5 <210> SEQ ID NO108 <211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: synthetic <400> SEQUENCE: 108 Gly Asn Gln Lys Cys Leu Gln HisAsn Arg Cys Ser Thr 1 5 10 <210> SEQ ID NO 109 <211> LENGTH: 9 <212>TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: synthetic <400>SEQUENCE: 109 Ser Gln Thr Trp Ser Phe Pro Glu His 1 5 <210> SEQ ID NO110 <211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: synthetic <400> SEQUENCE: 110 Glu Pro Met Ala Arg Pro Trp GluArg Lys Gln Asp Arg 1 5 10 <210> SEQ ID NO 111 <211> LENGTH: 13 <212>TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: synthetic <400>SEQUENCE: 111 Gly Leu Thr Ser Ala Thr Arg Pro Tyr Gly Arg Gln Trp 1 5 10<210> SEQ ID NO 112 <211> LENGTH: 22 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <221> NAME/KEY: UNSURE <222>LOCATION: (7) <223> OTHER INFORMATION: The x at position 7 representseither Glutamic acid or Aspartic acid. <221> NAME/KEY: UNSURE <222>LOCATION: (8)..(15) <223> OTHER INFORMATION: The x at positions 8through 15 represents any amino acid. <221> NAME/KEY: UNSURE <222>LOCATION: (22) <223> OTHER INFORMATION: The x at position 22 representseither Glutamic acid or Aspartic acid. <223> OTHER INFORMATION:Description of Artificial Sequence: synthetic <400> SEQUENCE: 112 SerLys Val Ile Leu Phe Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser 1 5 10 15Lys Val Ile Leu Phe Xaa 20 <210> SEQ ID NO 113 <211> LENGTH: 4 <212>TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: synthetic <400>SEQUENCE: 113 Tyr Trp Asp Trp 1 <210> SEQ ID NO 114 <211> LENGTH: 4<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221>NAME/KEY: UNSURE <222> LOCATION: (2) <223> OTHER INFORMATION: The x atposition 2 represents either Aspartic acid or Glutamic acid. <223> OTHERINFORMATION: Description of Artificial Sequence: synthetic <400>SEQUENCE: 114 Trp Xaa Tyr Tyr 1 <210> SEQ ID NO 115 <211> LENGTH: 20<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: synthetic <400>SEQUENCE: 115 Cys Cys Cys Thr Cys Ala Thr Ala Gly Thr Thr Ala Gly CysGly Thr 1 5 10 15 Ala Ala Cys Gly 20 <210> SEQ ID NO 116 <211> LENGTH:12 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:<221> NAME/KEY: UNSURE <222> LOCATION: (1) <223> OTHER INFORMATION: Thexaa at position 1 represents either Lysine, Asparagine or Threonine.<221> NAME/KEY: UNSURE <222> LOCATION: (2) <223> OTHER INFORMATION: Thexaa at position 2 represents either Histidine, Proline or Asparagine.<221> NAME/KEY: UNSURE <222> LOCATION: (3) <223> OTHER INFORMATION: Thexaa at position 3 represents either Alanine or Valine. <221> NAME/KEY:UNSURE <222> LOCATION: (4) <223> OTHER INFORMATION: The xaa at position4 represents either Histidine or Glutamine. <221> NAME/KEY: UNSURE <222>LOCATION: (8) <223> OTHER INFORMATION: The xaa at position 8 representseither Histidine or Glutamine. <221> NAME/KEY: UNSURE <222> LOCATION:(12) <223> OTHER INFORMATION: The xaa at position 12 represents eitherSerine or Threonine. <223> OTHER INFORMATION: Description of ArtificialSequence: synthetic <400> SEQUENCE: 116 Xaa Xaa Xaa Xaa Tyr Trp Thr XaaMet Phe Tyr Xaa 1 5 10 <210> SEQ ID NO 117 <211> LENGTH: 12 <212> TYPE:PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY:UNSURE <222> LOCATION: (1) <223> OTHER INFORMATION: The xaa at position1 represents either Isoleucine or Leucine. <221> NAME/KEY: UNSURE <222>LOCATION: (4) <223> OTHER INFORMATION: The xaa at position 4 representseither Proline or Arginine. <221> NAME/KEY: UNSURE <222> LOCATION: (10)<223> OTHER INFORMATION: The xaa at position 10 represents eitherProline or Arginine. <223> OTHER INFORMATION: Description of ArtificialSequence: synthetic <400> SEQUENCE: 117 Xaa Pro His Xaa Pro Met Tyr TrpThr Xaa Val Phe 1 5 10 <210> SEQ ID NO 118 <211> LENGTH: 13 <212> TYPE:PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY:UNSURE <222> LOCATION: (1) <223> OTHER INFORMATION: The xaa at position1 represents either Histidine or Glutamine. <221> NAME/KEY: UNSURE <222>LOCATION: (2) <223> OTHER INFORMATION: The xaa at position 2 representseither Glutamic acid or Glycine. <221> NAME/KEY: UNSURE <222> LOCATION:(4) <223> OTHER INFORMATION: The xaa at position 4 represents eitherAspartic acid or Glutamic acid. <221> NAME/KEY: UNSURE <222> LOCATION:(6) <223> OTHER INFORMATION: The xaa at position 6 represents eitherTyrosine or Asparagine. <221> NAME/KEY: UNSURE <222> LOCATION: (11)<223> OTHER INFORMATION: The xaa at position 11 represents eitherAlanine, Aspartic acid, Proline or Threonine. <223> OTHER INFORMATION:Description of Artificial Sequence: synthetic <400> SEQUENCE: 118 XaaXaa Trp Xaa Tyr Xaa Trp Asp Trp Thr Xaa Phe Trp 1 5 10 <210> SEQ ID NO119 <211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <221> NAME/KEY: UNSURE <222> LOCATION: (2) <223> OTHERINFORMATION: The xaa at position 2 represents either Histidine, Proline,Threonine or is optionally omitted. <221> NAME/KEY: UNSURE <222>LOCATION: (3) <223> OTHER INFORMATION: The xaa at position 3 representseither Histidine, Asparagine, Serine or Tyrosine. <221> NAME/KEY: UNSURE<222> LOCATION: (4) <223> OTHER INFORMATION: The xaa at position 4represents either Aspartic acid or Glutamic acid. <221> NAME/KEY: UNSURE<222> LOCATION: (5) <223> OTHER INFORMATION: The xaa at position 5represents either Phenyalanine or Tyrosine. <221> NAME/KEY: UNSURE <222>LOCATION: (6) <223> OTHER INFORMATION: The xaa at position 6 presentseither Aspartic acid or Glutamic acid. <221> NAME/KEY: UNSURE <222>LOCATION: (9) <223> OTHER INFORMATION: The xaa at position 9 representseither Aspartic acid or Glutamic acid. <221> NAME/KEY: UNSURE <222>LOCATION: (12) <223> OTHER INFORMATION: The xaa at position 12represents either Phenyalanine, Leucine, Methionine or Tyrosine. <223>OTHER INFORMATION: Description of Artificial Sequence: synthetic <400>SEQUENCE: 119 Tyr Xaa Xaa Xaa Xaa Xaa Trp Trp Xaa Tyr Tyr Xaa 1 5 10<210> SEQ ID NO 120 <211> LENGTH: 48 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: synthetic <400> SEQUENCE: 120 Gly Gly Gly SerLys Val Ile Leu Phe Glu Gly Pro Ala Gly Arg Thr 1 5 10 15 Ile Trp GluPro Lys Glu Ala Ser Asn His Thr Gly Ala Pro Gly Ser 20 25 30 Lys Val IleLeu Phe Glu Gly Gly Pro Gly His His His His His His 35 40 45 <210> SEQID NO 121 <211> LENGTH: 44 <212> TYPE: PRT <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: synthetic <400> SEQUENCE: 121 Gly Gly Gly Ser LysVal Ile Leu Phe Glu Gly Pro Ala Gly Arg Trp 1 5 10 15 Thr Trp Glu ProIle Ser Glu Gly Ala Pro Gly Ser Lys Val Ile Leu 20 25 30 Phe Glu Gly GlyPro Gly His His His His His His 35 40 <210> SEQ ID NO 122 <211> LENGTH:44 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence: synthetic<400> SEQUENCE: 122 Gly Gly Gly Ser Lys Val Ile Leu Phe Glu Gly Pro AlaGly Ser Gly 1 5 10 15 Ser Ala Gly Ser Gly Ala Ser Gly Ala Pro Gly SerLys Val Ile Leu 20 25 30 Phe Glu Gly Gly Pro Gly His His His His His His35 40

We claim:
 1. A complex comprising a fluorophore dye and a peptidecomprising a sequence of at least about 8 amino acids, wherein said dyeis bound by said peptide and wherein said peptide has an amino acidsequence consisting of SEQ ID No:
 15. 2. The complex according to claim1, wherein said complex has a dissociation constant less than or equalto about 0.5 micromolar.
 3. The complex according to claim 1, whereinsaid dye is Texas Red and said peptide has an amino acid sequenceconsisting of SEQ ID NO:15.
 4. A complex according to claim 1, whereinsaid fluorophore dye is selected from the group consisting of Texas Red,Rhodamine Red, Oregon Green 514, and Fluorescein.
 5. The complexaccording to claim 1, wherein said complex has an excitation spectrumthat differs from the excitation spectrum of said fluorophore dye in theabsence of said peptide.
 6. The complex according to claim 1 or 3,wherein said complex has an emission spectrum that differs from theemission spectrum of said fluorophore dye in the absence of saidpeptide.
 7. A method of binding a peptide to a fluorophore dye,comprising; contacting said fluorophore dye with said peptide, whereinsaid peptide has an amino acid sequence consisting of SEQ ID NO:15 thatbinds said fluorophore dye.
 8. The method according to claim 7, whereinsaid binding alters the emission spectrum of said fluorophore dye. 9.The method according to claim 7 or 8, wherein said binding alters theexcitation spectrum of said fluorophore dye.
 10. The method according toclaim 7, wherein said peptide is fused to a protein.